U.S. patent application number 10/850359 was filed with the patent office on 2005-12-01 for methods of human leukocyte antigen typing by neighboring single nucleotide polymorphism haplotypes.
Invention is credited to Lander, Eric S., Rioux, John D., Walsh, Emily.
Application Number | 20050266410 10/850359 |
Document ID | / |
Family ID | 35425773 |
Filed Date | 2005-12-01 |
United States Patent
Application |
20050266410 |
Kind Code |
A1 |
Walsh, Emily ; et
al. |
December 1, 2005 |
Methods of Human Leukocyte Antigen typing by neighboring single
nucleotide polymorphism haplotypes
Abstract
The disclosure relates to novel approaches to mapping the MHC
region and provides novel methods of genotyping the HLA loci. A
haplotype map of the region and methods of using the map are also
disclosed.
Inventors: |
Walsh, Emily; (Cambridge,
MA) ; Rioux, John D.; (Cambridge, MA) ;
Lander, Eric S.; (Cambridge, MA) |
Correspondence
Address: |
FISH & NEAVE IP GROUP
ROPES & GRAY LLP
ONE INTERNATIONAL PLACE
BOSTON
MA
02110-2624
US
|
Family ID: |
35425773 |
Appl. No.: |
10/850359 |
Filed: |
May 19, 2004 |
Current U.S.
Class: |
435/6.11 ;
435/6.12; 702/20 |
Current CPC
Class: |
C12Q 2600/156 20130101;
C12Q 1/6881 20130101 |
Class at
Publication: |
435/006 ;
702/020 |
International
Class: |
C12Q 001/68; G06F
019/00; G01N 033/48; G01N 033/50 |
Goverment Interests
[0001] Work described herein was funded, in whole or in part, from
the National Cancer Institute, National Institutes of Health, under
contract N01-CO-12400. The United States government has certain
rights in the invention.
Claims
We claim:
1. An SNP-haplotype map of a 4-Mb MHC region, said map comprising
evenly spaced SNPs, genotyped HLA genes, TAP genes, and
microsatellites.
2. The SNP-haplotype map according to claim 1, wherein the SNPs are
spaced at approximately every 20 kb of said MHC region.
3. A method of determining the identity of an HLA allele, said
method comprising a) determining the nucleotide present at one or
more extra-genic SNP sites corresponding to the HLA allele to be
assessed; and b) identifying said HLA allele based on the
nucleotide identity determined in a).
4. The method according to claim 3, wherein the HLA allele is an
HLA-A allele.
5. The method according to claim 4, wherein said one ore more SNP
sites are selected from the group consisting of: rs2517862,
rs1655930, rs1616549, rs376253, rs1961135, rs2517706, rs2517701,
rs2517699, rs435766, rs410909, rs2394255, rs1264807, rs2530388,
rs356963, rs2286405, rs2240619, rs3129012, and rs259938.
6. The method according to claim 4 comprising determining the
nucleotide present at more than one SNP site selected from the
group consisting of: rs2517862, rs1655930, rs1616549, rs376253,
rs1961135, rs2517706, rs2517701, rs2517699, rs435766, rs410909,
rs2394255, rs1264807, rs2530388, rs356963, rs2286405, rs2240619,
rs3129012, and rs259938.
7. The method according to claim 3, wherein the HLA allele is an
HLA-DRB1 allele.
8. The method according to claim 7, wherein said one ore more SNP
sites are selected from the group consisting of: rs742697,
rs523627, rs3129960, rs2395163, rs2395165, rs983561, rs2239804,
rs2213584, rs2395182, and rs2858860.
9. The method according to claim 8 comprising additionally
determining the nucleotide present at one or more SNP sites
selected from the group consisting of rs3129907, rs1059544, and
rs1987529.
10. The method according to claim 7 comprising determining the
nucleotide present at more than one SNP site selected from the
group consisting of: rs742697, rs523627, rs3129960, rs2395163,
rs2395165, rs983561, rs2239804, rs2213584, rs2395182, rs2858860
rs3129907, rs1059544, and rs1987529.
11. A method of predicting the likelihood of development of an
MHC-linked disease or an autoimmune disease in a human, comprising
determining the identity of an HLA allele in the human by
determining the nucleotide present at one or more extra-genic SNP
sites corresponding to the HLA allele to be assessed, wherein if
the HLA allele in the human is associated with an MHC-linked
disease or an autoimmune disease, the human has a greater
likelihood of development of said disease.
12. A method of predicting the likelihood of development of a
host-graft response in a human host, comprising determining the
identity of an HLA allele of the graft by determining the
nucleotide present at one or more extra-genic SNP sites
corresponding to the HLA allele to be assessed in the graft,
wherein if the HLA allele in the human host is identical to the
corresponding HLA allele in the graft, there is a low likelihood of
development of a host-graft response in the human host.
13. The method according to claim 12, optionally comprising
additionally determining the identity of the corresponding HLA
allele of the human host by determining the nucleotide present at
one or more extra-genic SNP sites corresponding to the HLA allele
to be assessed in the human host.
14. The method according to claim 12, comprising determining the
HLA-A allele in the graft by determining the nucleotide present at
one or more SNP sites selected from the group consisting of:
rs2517862, rs1655930, rs1616549, rs376253, rs1961135, rs2517706,
rs2517701, rs2517699, rs435766, rs410909, rs2394255, rs1264807,
rs2530388, rs356963, rs2286405, rs2240619, rs3129012, and
rs259938.
15. The method according to claim 12, comprising determining the
HLA-DRB1 allele in the graft by determining the nucleotide present
at one or more SNP sites selected from the group consisting of:
rs742697, rs523627, rs3129960, rs2395163, rs2395165, rs983561,
rs2239804, rs2213584, rs2395182, rs2858860 rs3129907, rs1059544,
and rs1987529.
16. A method of predicting the likelihood of development of a
host-graft response in a human host, comprising determining the
identity of an HLA allele of the graft by determining the
nucleotide present at one or more extra-genic SNP sites
corresponding to the HLA allele to be assessed in the graft,
wherein if the HLA allele in the human host is different from the
corresponding HLA allele in the graft, there is a high likelihood
of developing a host-graft response in the human host.
17. The method according to claim 16, optionally comprising
additionally determining the identity of the corresponding HLA
allele of the human host by determining the nucleotide present at
one or more extra-genic SNP sites corresponding to the HLA allele
to be assessed in the human host.
18. The method according to claim 16, comprising determining the
HLA-A allele in the graft by determining the nucleotide present at
one or more SNP sites selected from the group consisting of:
rs2517862, rs1655930, rs1616549, rs376253, rs1961135, rs2517706,
rs2517701, rs2517699, rs435766, rs410909, rs2394255, rs1264807,
rs2530388, rs356963, rs2286405, rs2240619, rs3129012, and
rs259938.
19. The method according to claim 16, comprising determining the
HLA-DRB1 allele in the graft by determining the nucleotide present
at one or more SNP sites selected from the group consisting of:
rs742697, rs523627, rs3129960, rs2395163, rs2395165, rs983561,
rs2239804, rs2213584, rs2395182, rs2858860 rs3129907, rs1059544,
and rs1987529.
Description
BACKGROUND OF THE INVENTION
[0002] The classical Human Leukocyte Antigen (HLA) loci are the
most highly variable genes in the human genome. Historically,
attempts to characterize the region have focused on a handful of
highly variable, classical HLA genes (class-I genes: HLA-A, HLA-B,
and HLA-C; and class-II genes: HLA-DRB1, HLA-DQA1, HLA-DQB1,
HLA-DPA1, and HLA-DPB1). These genes encode antigen-presenting
molecules that mediate acquired immune response during infection,
as well as host-graft responses after organ transplantation. All
organ transplant donors and recipients are typed for these genes in
order to best match donor with recipient. Also, these genes have
been associated with many human autoimmune and inflammatory
diseases, and many research laboratories genotype their human
subjects for these loci as a matter of course. The HLA loci were
originally studied by lower resolution serotyping techniques until
the recent advent of "dot blot" hybridization-based molecular
typing such as SSOP and SSP (Dynal Biotech, Biotest, One Lambda)
that greatly improved examination of the region. Direct sequencing
of HLA alleles is also possible. However, these current methods are
laborious and expensive. Accordingly, novel approaches to map the
HLA loci in the context of the MHC region are desirable.
SUMMARY OF THE INVENTION
[0003] Accordingly, the invention provides a more uniform,
comprehensive map of commonly linked variation, e.g., a haplotype
map, that will help to discriminate between causal alleles and
variation that is merely in linkage disequilibrium (LD) with them.
Such a resource will also allow a more complete description of the
haplotype structure and, potentially, insight into the evolutionary
and recombinational history of the chromosomal region in
question.
[0004] The invention provides an integrated SNP-haplotype map of a
4-Mb major histocompatibility complex (MHC) region. Preferably, the
integrated map comprises SNPs that are preferred to be reliable,
polymorphic, and evenly spaced, e.g., one SNP every 20 kb. The
integrated map further comprises genotyped HLA genes, TAP genes,
microsatellites, or combination thereof.
[0005] The invention further features a novel method of genotyping
Human Leukocyte Antigen (HLA) genes using patterns of neighboring
single nucleotide polymorphisms (SNPs). The SNP-based method is an
improvement over existing hybridization-based techniques, as it
allows quick and inexpensive genotyping of the HLA loci. This
method does not directly assess the intra-gene variation, as is
done by all other current methods for HLA genotyping, but rather
define HLA genotypes by studying the neighboring extra-genic
variation(s) which falls outside the HLA allele to be genotyped and
which, due to LD patterns, is conveniently linked to the HLA loci.
Identification of the correlation of this extra-genic variation to
the HLA gene alleles allows for the discovery and utilization of
surrogate markers for HLA genotypes.
[0006] This approach to genotype the HLA loci overcomes a
substantial technical difficulty to applying high-throughput
genotyping techniques to these hypervariable genes. By focusing on
variation outside of the hypervariable HLA genes themselves, this
method avoids the pitfalls of polymerase chain reaction (PCR)
primer design in locations where nucleotide diversity can be as
high as 12% (i.e., an average of 12 base pairs substituted per 100
nucleotides assessed). Instead, ancestral "hitchhiking mutations"
outside of these genes are used to resolve HLA genotypes with
traditional SNP genotyping methods. This approach can be employed
to map variation(s) in the regions neighboring HLA genes to fully
resolve all known common HLA gene variants in multiple different
ethnic populations. This method can benefit clinical laboratories
typing individuals for transplantation procedures, as well as
research laboratories that are interested in studying HLA gene
variation(s) in particular patient populations or disease
associations. Further, this method can be employed to predict the
likelihood or probability of developing a disease, particularly
MHC-linked diseases or autoimmune diseases. Alternatively, this
method can be employed to predict the likelihood or probability of
developing an immune response, e.g., a response against infection
or a host-graft response (e.g., elicited by organ transplantation)
in a subject, preferably a human subject.
[0007] One aspect of the invention provides a method of genotyping
an HLA gene, such as for example an HLA-A or an HLA-DRB1 gene. The
method comprises determining the nucleotide present at one or more
extra-genic SNP sites, wherein the SNP is associated with an HLA
genotype. The extra-genic SNP sites correspond to the HLA allele to
be genotyped, that is, the SNP sites are outside and in the
neighboring region(s) of the HLA allele to be genotyped. For
example, to genotype the HLA-A allele, an extra-genic SNP to be
assessed that corresponds to the HLA-A allele can be rs2517862,
rs1655930, rs1616549, rs376253, rs1961135, rs2517706, rs2517701,
rs2517699, rs435766, rs410909, rs2394255, rs1264807, rs2530388,
rs356963, rs2286405, rs2240619, rs3129012, rs259938, or any
combination thereof. Another example involves genotyping the
HLA-DRB1 allele, wherein an extra-genic SNP to be assessed can be
rs742697, rs523627, rs3129960, rs2395163, rs2395165, rs983561,
rs2239804, rs2213584, rs2395182, rs2858860, rs3129907, rs1059544,
rs1987529, or any combination thereof.
[0008] Another aspect of the invention provides a method of
predicting or assisting in predicting the likelihood of developing
a disease, in particular an inflammatory disease, an MHC-linked
disease, or an autoimmune disease, in a subject, preferably a human
subject. The method comprises genotyping an HLA gene in the subject
to be tested by determining the nucleotide present at one or more
extra-genic SNP sites, wherein the SNP is associated with the HLA
genotype.
[0009] A further aspect of the invention provides a method of
predicting or assisting in predicting the likelihood of developing
an immune response in a subject, preferably a human subject. An
immune response may be developed against an infection or
inflammation. Alternatively, an immune response may comprise a
host-graft response, e.g., rejection of organ transplants. The
method comprises genotyping an HLA gene in the subject to be tested
by determining the nucleotide present at one or more extra-genic
SNP sites, wherein the SNP is associated with an HLA genotype.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
[0011] FIG. 1A-1E shows an integrated SNP map of the 4-Mb MHC in
CEPH Europeans. FIG. 1A shows the location and exon-intron
structure for a subset of genes above the map, for positional
reference. FIG. 1B shows 201 reliable, polymorphic SNPs, indicated
on the map with ticks below the line. Ticks above the line are
placed with 100-kb spacing. FIG. 1C shows haplotype blocks below
and common haplotype variants (13% frequency) shown as colored
lines (thickness indicates relative population frequency). Colors
serve only to distinguish haplotypes and do not indicate block
to-block connections. Asterisks are found below the seven largest
haplotype blocks. FIG. 1D shows pairwise D' values (Lewontin 1964)
for SNPs indicated below the haplotype blocks. Note that each block
represents a single D' calculation and is placed in the middle
between the two SNPs analyzed. Red indicates strong LD and high
confidence of the D' estimate (D'>0.95; LOD>=3.0). Blue
indicates strong LD with low confidence of the estimate of D'
(D'=1; LOD<3.0). White indicates weak LD. FIG. 1E, shows the
relative recombination rate, which is based on the sperm meiotic
map, indicated in bar-graph form, where the value on the line is
the regional average, 0.49 cM/Mb. Green bars indicate recombination
rates >0.49 cM/Mb, and yellow bars indicate rates <0.49
cM/Mb. The black arrowhead denotes a region of well-mapped
recombination rate from Jeffreys et al. (2001). SNP marker density
in that region is too low to comment on any similarities between
the studies described herein. Note that five of seven long
haplotype blocks map to regions where the recombination rate is
=<0.49 cM/Mb. The remaining two long blocks are found in domains
where recombination rates are 0.64 cM/Mb and 0.83 cM/Mb (rates near
or below the genomewide average).
[0012] FIG. 2A-2D show block comparison between the MHC and other
autosomal regions. FIG. 2A shows a plot of LD by physical distance
revealing that LD is extended in the MHC. FIG. 2B shows that the
average physical length of blocks in the MHC is longer than in the
rest of the genome. FIG. 2C shows that, measured by genetic
distance, block size in the MHC is somewhat less than in the rest
of the genome. FIG. 2D shows that the number of haplotype variants
in blocks not spanning classical HLA genes is the same as elsewhere
in the genome.
[0013] FIG. 3A-3C shows EHH analysis of haplotype blocks,
microsatellites, HLA genes, and TAP genes in the region. EHH is
computed as the percentage of instances in which two randomly
selected chromosomes with the same variant locus have identical
alleles at all SNPs assayed up to a particular distance from that
locus (e.g., an EHH of 0.5 at marker X means that 50% of possible
pairings of a particular variant exhibit sequence identity from the
locus to marker X). FIG. 3A shows points representing the EHH at a
distance of 0.25 cM from an allele at a particular locus. Outlying
variants are indicated in color. The nine outlying variants define
three extended haplotypes. The six points labeled as "1" indicate
variants that map on the DRB1*1501 haplotype (associated with lupus
and MS). The two overlapping points labeled as "2" indicate
variants C*0701 and D6S2840*219, which are both found on a
haplotype associated with autoimmune diabetes, lupus, and
hepatitis. The point labeled as "3" indicates DRB1*1101 (associated
with pemphigoid disease). FIG. 3B shows a
recombination-distance-based map of the region.
Microsatellites/genes are labeled and indicated with ticks above
the line. FIG. 3C shows EHH values for loci that have at least one
outlying variant. Outlying variants were seen at 7 of the 48
independent loci tested. The X-axis denotes distance in cM. EHH
values are converted to grayscale values: EHH of 1 p black, EHH of
0.5 p 50% grayscale. The solid lines 4-10 indicate the locus about
which values were derived. The dotted lines 11-17 and 11'-17'
indicate 0.25-cM distance at which outliers were assessed. Two
HLA-C alleles, C*0702 and C*0701, are extended, as are two DRB1
alleles, DRB1*1501 and DRB1*1101. The other HLA gene alleles with
extended haplotypes are DQA1*0102 and DQB1*0602. The microsatellite
alleles with extended haplotypes are D6S2793*244, D6S2876*11, and
D6S2840*219.
[0014] FIG. 4A-4B show correlation of HLA alleles to SNP haplotype
background. A map of region showing placement of SNPs and
haplotypes assayed is shown for reference. Multi-SNP haplotypes are
coded by single capital letters. FIG. 4A shows SNP-HLA haplotypes
sorted by HLA allele. Percents indicate the percentage of a
particular HLA allele that falls on the indicated SNP haplotype.
FIG. 4B shows SNP-HLA haplotypes sorted by SNP haplotype allele.
Percents indicate the percentage of a particular SNP haplotype
allele that bears the indicated HLA allele. Counts are overall
number of chromosomes bearing the SNP-HLA haplotype indicated.
DETAILED DESCRIPTION OF THE INVENTION
[0015] Overview
[0016] In order to fully map the variation, especially variations
associated with diseases, in the MHC region, an SNP haplotype map
of the region was created. To be able to integrate this map with
the wealth of findings from association studies, 201 reliable,
polymorphic, evenly spaced SNPs (target density: one SNP every 20
kb) were genotyped in 136 independent chromosomes also genotyped
for nine HLA genes, two TAP genes (involved in antigen processing),
and 18 microsatellites. Markers were genotyped in families (18
multigenerational European pedigrees) to allow direct assessment of
chromosomal phase and, thus, simple reconstruction of haplotypes.
Using these SNP data, the haplotype patterns of the region and
mapped these patterns were examined, relative to both genetic and
physical distance, as assayed by an exceedingly high-resolution
recombination map (FIGS. 1A-1E). This recombination map is the
result of the analysis of 20,000 sperm meioses from 12 men (Cullen
et al. 2002).
[0017] This SNP density is a large first step toward a
comprehensive characterization of the patterns of common variation
in the MHC. Here, this map is used to first explore the structure
of LD in the region, with respect to both haplotype blocks and
extended haplotypes. Next, SNP-haplotype variation in the MHC was
examined, first considering regions between the classical HLA loci
and then examining SNP-haplotype variation across these genes. The
question of whether the SNP haplotype diversity near classical HLA
loci contained enough information to predict the HLA allele carried
on the chromosome was also examined.
[0018] FIGS. 1A-1E show an integrated map of the SNP,
microsatellite, and HLA variation in the MHC. This map shows that,
aside from the classical HLA loci, the variation and LD structure
of the MHC are not different from a genomewide control data set.
Specifically, whereas LD appears to extend over longer physical
distances in the MHC, this seems to be accounted for by the reduced
recombination rate in the region. Furthermore, this map shows that,
in the regions that do not span classical HLA loci, the number of
common haplotype alleles in the MHC are not different from the rest
of the genome.
[0019] The integrated map of FIGS. 1A-1E and the results shown in
FIGS. 3A-3C and 4A-4B demonstrate that multiblock SNP haplotypes
contain considerable predictive information for common HLA alleles
at HLA-A, HLA-B, HLA-C, and HLA-DRB1. Multiblock SNP haplotypes
should enable cost-efficient, large-scale exploration of the
variation at the classical HLA loci and beyond. An additional
implication of these results is that multiblock SNP haplotypes may
be sufficient to identify low-frequency variants throughout the
genome. Such low-frequency variants would likely be missed in
single, block-based, common variant analysis; however, their
contribution to disease may be assayed by use of multiblock
haplotypes in analysis.
[0020] This integrated variation map of the MHC has considerable
utility. In the 50 years of study since its first discovery, the
MHC has been implicated in almost every human inflammatory and
autoimmune disease. Although the MHC has been studied by typing of
the classical HLA genes and microsatellites for many years, only
rarely has this analysis definitively identified causal variation.
Often, association studies using these methods implicate more than
one allele at a single locus as influencing disease susceptibility.
Although this may represent allelic heterogeneity underlying
disease, reinterpreting such results with attention to shared
SNP-haplotype variation might point to additional hypotheses
regarding the causal variant. For instance, one may find that two
different disease-predisposing HLA alleles share a common SNP
haplotype, which suggests that a variant carried on that haplotype
may, in fact, be the underlying cause of disease. Another common
finding in MHC studies is that an extended haplotype, rather than a
single variant, is associated with disease. A uniform map of the
variation in the region would allow fine mapping of association
signals on the basis of rare recombinant chromosomes. Because SNPs
are more abundantly present, reliably typed, and cost efficient
than microsatellites, they are an excellent choice for this sort of
large-scale, high density genotyping. A denser sampling of all the
haplotype variation in the region will allow researchers to fully
consider all of the 120 genes that lie in the MHC, rather than to
focus solely on the classical HLA loci.
[0021] The map as shown in FIGS. 1A-1E identifies haplotype blocks
covering 24.5% of the MHC. On the basis of the estimated average
size of blocks in this region, SNP coverage must be increased
four-fold to reach saturation. This map in FIGS. 1A-1E is based on
the genotypes only of individuals of European ancestry; variation
in other populations must also be examined to unify MHC association
results between populations. The additional SNPs to be discovered,
as well as genotype information in other populations, may be
employed to build a more complete map according to the materials
and methods described herein that were employed to build the map as
shown in FIGS. 1A-1E.
[0022] Ultimately, a full understanding of the patterns of LD and
haplotype diversity of this region should allow the identification
of a subset of SNPs required for disease studies. This will allow
MHC-association studies to be completed cost effectively by using a
combination of haplotype-tagging and HLA allele-tagging SNPs.
Although a large number of SNPs were used to construct the map as
shown in FIGS. 1A-1E, and more SNPs will be needed to fully
describe the haplotype structure of the region, an estimate of
10-15 SNPs per locus may be sufficient for common, classical HLA
alleles. Moreover, in cases where there is already significant
association to a particular locus, these informative SNPs may be
used to map outward from the original signal and delimit the region
of association. An estimate of a few dozen SNPs may be needed in
such endeavors.
[0023] SNP-based haplotype approaches will allow the examination of
larger disease cohorts and enable the identification of rare
recombinant haplotypes that would refine association signals and
potentially identify the causal alleles for MHC-associated
diseases.
[0024] Integrated Map
[0025] SNPs used in creating the integrated map as shown in FIG. 1
include the following SNPs, as shown in TABLE 1. All SNPs are
located on human chromosome 6, and their respective chromosome
positions are shown in Column CHROM_POS. The frequencies of allele
types are also shown in Columns FREQ1 and FREQ2. The primer
sequences, as well as probe information and flanking sequences for
the SNPs are described in detail at:
http://www.broad.mit.edu/mpg/idrg/projects/HLA_data/SNP_Info.xls
(incorporated herein by reference in its entirety). The primer
sequences for SNPs are also provided herein in TABLE 2.
1TABLE 1 SNP CHROM CHROM_POS ALLELE1 FREQ1 ALLELE2 FREQ2 rs1611750
6 29891606 G 0.187969925 T 0.812030075 rs2517862 6 29898525 A
0.192592593 A 0.807407407 rs1655930 6 29942211 A 0.821705426 C
0.178294574 rs2517706 6 29963094 C 0.602941176 T 0.397058824
rs435766 6 29983284 A 0.352941176 G 0.647058824 rs410909 6 29992085
A 0.098484848 C 0.901515152 rs1264807 6 30002241 A 0.801470588 C
0.198529412 rs356963 6 30013075 C 0.925925926 G 0.074074074
rs3129012 6 30032096 A 0.189393939 G 0.810606061 rs259938 6
30051799 C 0.701492537 G 0.298507463 rs2844800 6 30061564 A
0.921875 G 0.078125 rs3132129 6 30071689 A 0.073529412 G
0.926470588 rs1150736 6 30098575 A 0.294117647 G 0.705882353
rs1264709 6 30112291 A 0.801470588 T 0.198529412 rs1264701 6
30122157 G 0.860294118 T 0.139705882 rs2427749 6 30163400 C
0.820895522 T 0.179104478 rs1015465 6 30170856 C 0.110294118 T
0.889705882 rs1573297 6 30200857 C 0.694656489 T 0.305343511
rs2074477 6 30216292 A 0.111111111 G 0.888888889 rs2844786 6
30228928 A 0.333333333 C 0.666666667 rs2074473 6 30238687 C
0.485294118 T 0.514705882 rs2517614 6 30248435 A 0.169117647 G
0.830882353 rs2021722 6 30258150 A 0.167938931 G 0.832061069
rs885916 6 30260107 A 0.140740741 G 0.859259259 rs928824 6 30280200
A 0.087301587 G 0.912698413 rs968909 6 30300336 C 0.838235294 T
0.161764706 rs1264626 6 30303763 A 0.825396825 G 0.174603175
rs261945 6 30327954 C 0.544776119 G 0.455223881 rs3094628 6
30341046 C 0.141791045 G 0.858208955 rs1264582 6 30350326 A
0.081481481 G 0.918518519 rs1110464 6 30351625 A 0.529411765 G
0.470588235 rs1264579 6 30358950 C 0.903703704 G 0.096296296
rs3094054 6 30389245 G 0.846153846 T 0.153846154 rs3129820 6
30399307 A 0.141791045 G 0.858208955 rs970270 6 30402909 C
0.768595041 T 0.231404959 rs2187978 6 30418709 C 0.888888889 T
0.111111111 rs1264562 6 30428292 A 0.362204724 C 0.637795276
rs1150769 6 30438720 A 0.438461538 G 0.561538462 rs1264511 6
30469758 C 0.571428571 G 0.428571429 rs2524172 6 30489460 A
0.125925926 G 0.874074074 rs2021720 6 30504972 C 0.410852713 G
0.589147287 rs1059510 6 30513496 C 0.669230769 T 0.330769231
rs2844724 6 30524708 C 0.348484848 T 0.651515152 rs2516650 6
30550400 C 0.179104478 G 0.820895522 rs1362119 6 30555226 A
0.656716418 T 0.343283582 rs1468079 6 30562131 A 0.338461538 C
0.661538462 rs2516640 6 30572208 C 0.121212121 T 0.878787879
rs2074505 6 30576689 C 0.351145038 T 0.648854962 rs3130242 6
30593090 A 0.654411765 G 0.345588235 rs1264440 6 30606834 C
0.669117647 T 0.330882353 rs2252745 6 30635057 C 0.325925926 T
0.674074074 rs1059612 6 30764464 C 0.858208955 T 0.141791045
rs3129973 6 30776893 C 0.851851852 T 0.148148148 rs3130673 6
30802269 G 0.843283582 T 0.156716418 rs1264377 6 30820115 C
0.828358209 T 0.171641791 rs1264352 6 30845173 C 0.172932331 G
0.827067669 rs2535335 6 30868020 A 0.7 G 0.3 rs3095354 6 30891957 C
0.544117647 T 0.455882353 rs1264332 6 30901222 C 0.715384615 G
0.284615385 rs1264314 6 30926804 A 0.782945736 T 0.217054264
rs1264297 6 30940639 A 0.705882353 C 0.294117647 rs2532936 6
30950044 G 0.298507463 T 0.701492537 rs3132571 6 30961148 A
0.632352941 G 0.367647059 rs2517434 6 30999873 C 0.92481203 T
0.07518797 rs2253417 6 31025801 A 0.088235294 G 0.911764706
rs1619376 6 31039164 A 0.213235294 G 0.786764706 rs2523897 6
31049548 A 0.15037594 G 0.84962406 rs2844670 6 31061565 A
0.792592593 G 0.207407407 rs2517523 6 31082273 A 0.635658915 G
0.364341085 rs2523882 6 31097806 C 0.75 T 0.25 rs2517407 6 31122306
C 0.287878788 T 0.712121212 rs1064190 6 31130758 G 0.488549618 T
0.511450382 rs1265099 6 31161025 A 0.679389313 G 0.320610687
rs1265114 6 31173035 C 0.661764706 T 0.338235294 rs915660 6
31199448 C 0.874074074 G 0.125925926 rs1265181 6 31211656 C
0.257575758 G 0.742424242 rs3132502 6 31239363 A 0.280701754 G
0.719298246 rs1793895 6 31247789 G 0.748148148 T 0.251851852
rs3134756 6 31269208 C 0.257352941 T 0.742647059 rs1793892 6
31275440 C 0.139705882 T 0.860294118 rs3130542 6 31286439 A
0.274074074 G 0.725925926 rs364415 6 31327348 C 0.80952381 T
0.19047619 rs3130690 6 31340260 G 0.832061069 T 0.167938931
rs2524227 6 31356125 A 0.353383459 G 0.646616541 rs2854008 6
31366306 A 0.291044776 G 0.708955224 rs2853996 6 31386696 C
0.821705426 T 0.178294574 hCV2995747 6 31393338 A 0.777777778 G
0.222222222 rs2301747 6 31425383 C 0.902985075 G 0.097014925
rs2256184 6 31434180 A 0.612403101 G 0.387596899 hCV2995705 6
31441607 C 0.066176471 G 0.933823529 rs2855804 6 31521290 C
0.681481481 T 0.318518519 rs3132464 6 31531399 C 0.259259259 T
0.740740741 rs2516400 6 31553393 C 0.671641791 T 0.328358209
hCV3273612 6 31564553 A 0.674242424 T 0.325757576 rs361525 6
31606263 A 0.068181818 G 0.931818182 rs986475 6 31619673 C
0.066666667 T 0.933333333 rs2857595 6 31631860 A 0.161764706 G
0.838235294 rs2844476 6 31645038 A 0.378787879 G 0.621212121
rs750332 6 31670185 C 0.233082707 T 0.766917293 rs1052486 6
31673871 C 0.465648855 T 0.534351145 rs3117583 6 31682962 A
0.857142857 G 0.142857143 rs805256 6 31698902 A 0.803149606 G
0.196850394 rs805290 6 31711788 C 0.735294118 T 0.264705882
rs805281 6 31724677 A 0.736842105 G 0.263157895 rs805292 6 31753147
A 0.21641791 G 0.78358209 rs1150793 6 31789133 A 0.961538462 G
0.038461538 rs707928 6 31814030 A 0.681481481 G 0.318518519
rs480092 6 31836340 C 0.174242424 T 0.825757576 rs2075799 6
31850226 C 0.941176471 T 0.058823529 rs539689 6 31857089 C
0.492647059 G 0.507352941 rs2763979 6 31866094 C 0.653846154 T
0.346153846 rs3130679 6 31879245 A 0.904 G 0.096 rs574914 6
31890829 A 0.151515152 G 0.848484848 rs660550 6 31909117 A
0.52238806 C 0.47761194 rs605203 6 31918651 G 0.351145038 T
0.648854962 rs589428 6 31919809 G 0.62962963 T 0.37037037 rs558702
6 31941915 A 0.082706767 G 0.917293233 rs419788 6 31990590 C
0.705882353 T 0.294117647 rs429608 6 31992054 A 0.112781955 G
0.887218045 rs433061 6 32043622 A 0.090225564 G 0.909774436
rs1269852 6 32119789 C 0.080882353 G 0.919117647 rs2269425 6
32150480 C 0.823076923 T 0.176923077 rs204989 6 32188886 A
0.110294118 G 0.889705882 rs2071280 6 32191654 C 0.294117647 G
0.705882353 rs2071277 6 32210496 C 0.435114504 T 0.564885496
rs3130316 6 32250273 C 0.691588785 T 0.308411215 rs926070 6
32283275 C 0.346153846 T 0.653846154 rs742697 6 32318423 C
0.345864662 T 0.654135338 rs3129960 6 32327712 A 0.227941176 G
0.772058824 rs2022534 6 32333790 A 0.596899225 G 0.403100775
rs3129907 6 32350292 A 0.759398496 G 0.240601504 rs2143462 6
32361583 C 0.860294118 T 0.139705882 rs1555115 6 32381050 C
0.895522388 G 0.104477612 rs2395158 6 32401103 A 0.880597015 G
0.119402985 rs2395161 6 32414066 A 0.867647059 C 0.132352941
rs983561 6 32430210 A 0.785185185 C 0.214814815 rs2239802 6
32438402 C 0.723880597 G 0.276119403 rs7194 6 32439002 A
0.563492063 G 0.436507937 rs1987529 6 32502240 A 0.85 G 0.15
rs1059544 6 32578551 C 0.268292683 T 0.731707317 rs2858860 6
32598186 G 0.454545455 T 0.545454545 rs2395253 6 32717006 A
0.053030303 G 0.946969697 rs2857210 6 32738722 A 0.35483871 G
0.64516129 rs719654 6 32749097 A 0.22962963 G 0.77037037 rs2157080
6 32758398 A 0.376923077 G 0.623076923 rs2621343 6 32771751 C
0.383458647 T 0.616541353 rs1383267 6 32830393 C 0.560606061 T
0.439393939 rs1029295 6 32853431 C 0.105263158 T 0.894736842
rs241404 6 32862944 C 0.428571429 T 0.571428571 rs2187688 6
32868648 A 0.446153846 G 0.553846154 rs151719 6 32900606 A
0.785714286 G 0.214285714 rs188245 6 32955171 C 0.529411765 T
0.470588235 rs663310 6 33009016 C 0.217054264 T 0.782945736
rs2071351 6 33045675 A 0.84496124 G 0.15503876 rs2144014 6 33067793
C 0.703125 T 0.296875 rs3130216 6 33079418 A 0.574626866 G
0.425373134 rs3129272 6 33099767 C 0.696296296 T 0.303703704
rs2294478 6 33102058 A 0.474074074 C 0.525925926 rs734181 6
33133246 C 0.201550388 G 0.798449612 rs2076311 6 33148710 A
0.298507463 C 0.701492537 rs2855433 6 33161160 A 0.701492537 C
0.298507463 rs421446 6 33178124 A 0.729323308 G 0.270676692
rs213213 6 33186822 C 0.725925926 T 0.274074074 rs213194 6 33198941
A 0.234375 G 0.765625 rs105445 6 33220331 C 0.146153846 G
0.853846154 rs464865 6 33259080 A 0.441176471 G 0.558823529
rs1014779 6 33278611 A 0.533333333 G 0.466666667 rs1061783 6
33284571 A 0.544776119 G 0.455223881 rs3130267 6 33318929 G
0.5390625 T 0.4609375 rs456993 6 33360326 C 0.444444444 T
0.555555556 rs211457 6 33367887 C 0.873134328 T 0.126865672
rs1705003 6 33388001 A 0.888888889 G 0.111111111 rs2076775 6
33396500 C 0.634328358 G 0.365671642 rs453590 6 33405670 C
0.634328358 T 0.365671642 rs1755047 6 33433266 C 0.451851852 G
0.548148148 rs210190 6 33466198 A 0.066176471 G 0.933823529
rs1755038 6 33467280 A 0.909774436 G 0.090225564 rs769051 6
33476004 G 0.659090909 T 0.340909091 rs210180 6 33487120 A
0.348148148 T 0.651851852 rs210196 6 33509584 A 0.669117647 G
0.330882353 rs210203 6 33513090 A 0.37037037 G 0.62962963 rs210132
6 33538781 G 0.536764706 T 0.463235294 rs210135 6 33542803 A
0.827868852 T 0.172131148 rs210139 6 33545520 A 0.564885496 C
0.435114504 rs210145 6 33549551 C 0.467213115 G 0.532786885
rs396746 6 33558906 A 0.148148148 C 0.851851852 rs210120 6 33576523
A 0.588235294 G 0.411764706 rs407415 6 33581077 A 0.786764706 G
0.213235294 rs999943 6 33626118 C 0.266666667 T 0.733333333
rs2229634 6 33640290 C 0.701492537 T 0.298507463 rs658087 6
33667130 A 0.148148148 T 0.851851852 rs2281829 6 33677752 A
0.544117647 G 0.455882353 rs1555965 6 33679261 A 0.555555556 G
0.444444444 rs549652 6 33688213 A 0.147058824 G 0.852941176
rs608971 6 33703990 C 0.78030303 T 0.21969697 rs530614 6 33716891 A
0.161764706 G 0.838235294 rs2395449 6 33730616 A 0.388059701 T
0.611940299 rs943473 6 33745761 C 0.816176471 G 0.183823529
rs2395402 6 33755534 A 0.559701493 G 0.440298507 rs2894342 6
33776504 A 0.25 C 0.75 rs1547668 6 33777490 A 0.139344262 G
0.860655738
[0026]
2TABLE 2 snp HG12 Primer 1 (SEQ ID NOS: 1-639) Primer 2 (SEQ ID
NOS: 640-1278) rs1611750 29891606 ACGTTGGATGTGAGGACCACAAAAGTCAGG
ACGTTGGATGCCCATCAATTGACCCAGTTC rs2517862 29898525
ACGTTGGATGGGGAAAACAGCAAGGTACAG ACGTTGGATGTGTTCTTTCTCCCTTTGCAC
rs885933 29913724 ACGTTGGATGTGTAGCCAGTCATAGCTGTC
ACGTTGGATGACTTCTCAGCTGCATCGATG rs886399 29913789
ACGTTGGATGGCTATCTGCCCTTTTGCTAC ACGTTGGATGTGGCTACATTTGACACCCTC
rs2394233 29924799 ACGTTGGATGGATAAATGGGTGTTGTTTCG
ACGTTGGATGGCAAAACACGGAAAAAGTTC rs1054175 29925728
ACGTTGGATGGGCCCCATGATGTATAAATG ACGTTGGATGACAGGTACACTGCAAAAGTG
rs1611545 29931342 ACGTTGGATGACAATACCTGCAGTACCCTC
ACGTTGGATGAAAACTTCCCTCATCCCAGC rs1632910 29935874
ACGTTGGATGGGCTCAACAGACTCGGAATG ACGTTGGATGACGTGAGCATATGAGGGCAT
rs2517762 29938895 ACGTTGGATGTCCCTGGAATACTGATGAGG
ACGTTGGATGAAAGCAGAGAACAAGGCCTG rs1655930 29942211
ACGTTGGATGTGTAGTAATCCTAGTGCTGG ACGTTGGATGATGGGTCCAATTTTCCACCC
rs2905764 29953186 ACGTTGGATGTTTGGTGCCAGAGAGTAAGC
ACGTTGGATGTTCTGTCTCATGCACTCAGG rs1616549 29957706
ACGTTGGATGAGTTCACGTGGACATCCATG ACGTTGGATGTTTGTGCTGAAGTGTGCAGG
rs376253 29957969 ACGTTGGATGGGGTTATGGTGCATACGTTC
ACGTTGGATGTCACTCCAGGACTCAGGTTC rs1961135 29958142
ACGTTGGATGGAACCCTCCTTTTCAGTGAC ACGTTGGATGGGCTGATACTCTGGGTTATC
rs2735099 29958264 ACGTTGGATGGTCAGAAAAGATGGGCAGAC
ACGTTGGATGTGCTCCTCAATTCCACATGC rs2524037 29958386
ACGTTGGATGATAGGCTCCTTTGCAGAAGG ACGTTGGATGAAGAACCTTGGGACACGATG
rs2517706 29963094 ACGTTGGATGGGATTAGAAGCATGAGCCAC
ACGTTGGATGGGCACACAAGGTGCATTTTG rs2975041 29964111
ACGTTGGATGCTCCATTCTCTGTCTCAAAG ACGTTGGATGCTTGTATCTGACTGATTTTC
rs382875 29967813 ACGTTGGATGAGTCTTTGAGGGAAAGGAGG
ACGTTGGATGAAAATTCCTGGTGCCCAAGG rs2517701 29969404
ACGTTGGATGATTGGAGTCATGGGAACCTG ACGTTGGATGACCCTAGGTAAGAGGATGTG
rs2517699 29970029 ACGTTGGATGCCCCACTTCTCACATGATAC
ACGTTGGATGGCCTCTGTCTTCTCTTTCTG rs435766 29983284
ACGTTGGATGTCCATGCCTTTCTGTGTGGG ACGTTGGATGTGAGGAATAGGGGTCAGCAG
rs410909 29992085 ACGTTGGATGCACACAGATTCACACACACG
ACGTTGGATGAAGTCAGCCTGTCCCACAAC rs2246555 29992480
ACGTTGGATGATCTCCCCCCTTCTTCAGAG ACGTTGGATGGTCTCTTTTTCCTGGAGGTG
rs2394255 29993239 ACGTTGGATGGGTGTAAAGGAAACTGCAGG
ACGTTGGATGATGGAGACAGTCCTTTCCAG rs1264807 30002241
ACGTTGGATGTAACATTCCCCTTTCTCCAC ACGTTGGATGGAGATATATCTTACCCTAACC
rs1632926 30004710 ACGTTGGATGGCACAATTCTCATCCGACAC
ACGTTGGATGTAACCTCTGTCTCCTTCCAG rs2530388 30010564
ACGTTGGATGCCCAACTCTCAACAAGGTAG ACGTTGGATGTCAGCCTCGTTATTCCTTCC
rs356962 30012120 ACGTTGGATGAACACAGAAGGCAGAGGTTG
ACGTTGGATGAAAGTCTTGCTCTTGTCCCC rs356963 30013075
ACGTTGGATGTGTGGTTGCTCCATTCATGC ACGTTGGATGCAGACAAATGGCAGTTAGCC
rs2286405 30016829 ACGTTGGATGAAAACAGGCAGTGCATGAGC
ACGTTGGATGTCACCTCAAAGTTGCAAGCG rs2240619 30018890
ACGTTGGATGATTCCTCTCCGTCAGGACAG ACGTTGGATGATCTCCTGTAGATCTCCCGG
rs886997 30021056 ACGTTGGATGACAAGGTTCTACTGAAGGGC
ACGTTGGATGACCATGGGCTTTATGTGGTC rs3129012 30032096
ACGTTGGATGCCCACTTGGCATGGTGAATC ACGTTGGATGAAGGTCTTAGGAGAGGGCTG
rs1150743 30035516 ACGTTGGATGCTGGACATTTCATCAGGACC
ACGTTGGATGATCTCAGCATGTGAGGCTTC rs259938 30051799
ACGTTGGATGGAGGCTATGGTACCAAACTG ACGTTGGATGCTGTGGATTCTGGGATAGAG
rs2844800 30061564 ACGTTGGATGCTCGGACTCCTTTGCTCATT
ACGTTGGATGCCCACAGGAAAGGAGAAAAG rs259948 30065078
ACGTTGGATGACTTTCACTCCACTGCCTTC ACGTTGGATGAAACTTTCGTGCTGCAGGTG
rs3132129 30071689 ACGTTGGATGCGTCTCCCTTTGTAAGACAG
ACGTTGGATGACTGCTGAAGAGTGACAAGC rs1150736 30098575
ACGTTGGATGTCGCGGAGTTGTTGGTGGAAG ACGTTGGATGTAAACTTCCACAGGGCCTCC
rs1264709 30112291 ACGTTGGATGTTTTGGAGCTAGGATTCTGG
ACGTTGGATGCTACCTCACTTCTGCATTTC rs1264701 30122157
ACGTTGGATGCCCTCTATGCTCACTATCTC ACGTTGGATGAAAAGAGCCAAGGGCAACAC
rs2023472 30160044 ACGTTGGATGATTTTTTCAGGCTCCCTGTG
ACGTTGGATGTCAGTTTCTCAACCCAACCC rs2427749 30163400
ACGTTGGATGAGTGGGGTCACAATGTCTTC ACGTTGGATGTATTGTTTGAGCCTGGGAGG
rs1015465 30170856 ACGTTGGATGGATAGTGCCCATTCACACTG
ACGTTGGATGAATGTCCAGAGCTGATGAGG rs1419673 30181231
ACGTTGGATGAGTCATTGGCCTGTTTTTCG ACGTTGGATGTGACATCTACAAACAGTTTC
rs1362104 30186130 ACGTTGGATGGGGAAAAAAAACCGTAAGTG
ACGTTGGATGGGCAGTTGTAAATATTTTTC rs1573297 30200857
ACGTTGGATGAGTCTTGTGGCTGCAAGAAC ACGTTGGATGGGGAAGAATCTGCTTCCAAG
rs2285797 30204577 ACGTTGGATGATCCAAAGCACCCAAACCTG
ACGTTGGATGCAAAAGACACAGCTAAAGCC rs2074477 30216292
ACGTTGGATGTTCTTTGCACTCCACCTCTG ACGTTGGATGTGGATGTGTGGTAGTTCCTG
rs2844786 30228928 ACGTTGGATGTAGCCAAAGAAATCCTGAGC
ACGTTGGATGCCTGACAAAAATGTCTCTAG rs2074473 30238687
ACGTTGGATGACTTCCACTTCCCAGTAGAC ACGTTGGATGTGTACAAGAGTGCCTACCTG
rs2517614 30248435 ACGTTGGATGCATAAAAAGCTTCCACCAGC
ACGTTGGATGTCATCACTGCCATCACAAGC rs2021722 30258150
ACGTTGGATGACACTTGGCTTACTTTCCCC ACGTTGGATGAGCCCTGGTAGTTTTTGTGG
rs885916 30260107 ACGTTGGATGTGGATTTTTCTTCCCCACTC
ACGTTGGATGTAAGATGTTGCCACAGTTCC rs3129696 30270016
ACGTTGGATGAACTCCTGACGTGATCTGCC ACGTTGGATGAAAAAATAGGCTGGGCACGG
rs928824 30280200 ACGTTGGATGCGCAAAAAAAAGTTGCAGTC
ACGTTGGATGGGAATTGTTGGGTGATATGG rs3132656 30289505
ACGTTGGATGACCATGATTCTGAGGCCTCC ACGTTGGATGCTGCCGATAAAGACATACCC
rs968909 30300336 ACGTTGGATGCAGTGTGAAATTGGACCCTG
ACGTTGGATGTCCCTAAAGGGATCAATGGC rs1264626 30303763
ACGTTGGATGAGTCATGAAAGATCCACCCC ACGTTGGATGCCCACCCAAATTTCGTGTTC
rs261956 30320688 ACGTTGGATGTTAGCAGGTATGGTGGCATG
ACGTTGGATGAACTCCTGGCTCAAATGATC rs261945 30327954
ACGTTGGATGCATGGCCTCTTATGAGAACC ACGTTGGATGGGGCAACAAGAGTGAAACTG
rs3094628 30341046 ACGTTGGATGAGGTGTGTTGGAAGGTGGTG
ACGTTGGATGCCCATGCATGCAATTACCTC rs1264582 30350326
ACGTTGGATGTTGATTCCCCCTGCTGCTTC ACGTTGGATGTCGTGTCAGTGGAAGCTGGG
rs1110464 30351625 ACGTTGGATGCTCAGAACTGCTGAAAACTG
ACGTTGGATGAGACTCGTTGCTCTCTTTTC rs1264579 30358950
ACGTTGGATGTCTGCCTTCTTTGCTCAAGC ACGTTGGATGATTAGCTGAGTCTGGTGGTG
rs984801 30376931 ACGTTGGATGGCAAGTAGCAGGAAATTCAG
ACGTTGGATGCCTCTGGAAGATAAAATGGG rs3094054 30389245
ACGTTGGATGCCCAGTGGCAAATCAATTAC ACGTTGGATGAATAACCCCTGGCTCAGAAC
rs3129820 30399307 ACGTTGGATGCCTCCTAGTTTCTGCTTTCC
ACGTTGGATGCTCACAGAAGAGAGGATGAG rs970270 30402909
ACGTTGGATGTCAAAGGACTGCAGGAACAG ACGTTGGATGGCTGCAAATACATGTGTGGG
rs2187978 30418709 ACGTTGGATGAAATGTCAGAGTGGTGTGGG
ACGTTGGATGTGAGTGGGATTGAAAAGCTG rs1264562 30428292
ACGTTGGATGTGTCGCCTCCTGCACTTCAT ACGTTGGATGTCCAACAGACGCTTTTCTGG
rs1150769 30438720 ACGTTGGATGCCAGCAGTTCATTCCTGAAC
ACGTTGGATGTGGGCTGAGTTCCTCACTTG rs1264534 30449506
ACGTTGGATGTTCTTCTCTCTCTTCTTCTC ACGTTGGATGCGTTAATGAATCTAGGAGCTG
rs1264525 30459721 ACGTTGGATGGGAAACTATCACAAGGACAG
ACGTTGGATGCTCATTGTTCAGTTTCCACC rs1264511 30469758
ACGTTGGATGTATGATTCCCCTCCTCCTTC ACGTTGGATGGAACTATGATCCTGACCCTG
rs2187975 30480842 ACGTTGGATGTGCCATGATGGTAAGCTTCC
ACGTTGGATGTCTGCAGGCTTTATGGGAAG rs2524172 30489460
ACGTTGGATGACTTCCCTTTCTTAGCCACC ACGTTGGATGCACTGGGAGAGATGAGTATG
rs3131112 30503440 ACGTTGGATGCCTGGAGCTTTATAGTAAGTC
ACGTTGGATGGAAGGACTTTGAATATCCAC rs2021720 30504972
ACGTTGGATGAGGTCCTTGATTCTGGACTC ACGTTGGATGTTTCGCGCTGGGGAGCCTCT
rs2021719 30505249 ACGTTGGATGAGAATCCTGGACTCTCAAGG
ACGTTGGATGATTTGTCCCCAATCCATCGG rs1059510 30513496
ACGTTGGATGGGGACACCGCACAGATTTTC ACGTTGGATGCCTCGCTCTGATTGTAGTAG
rs2516665 30523713 ACGTTGGATGTTGAGACCATCCTGGCTAAC
ACGTTGGATGCCACCACGCCCAGAAAATTT rs2844724 30524708
ACGTTGGATGGTGAGATCAAGAGTACTATTC ACGTTGGATGCTGTGTCACTTGGTAAGTAG
rs2023608 30531495 ACGTTGGATGGGTTGTGCTAACCTGACTTC
ACGTTGGATGGTGGGAAGGATTCCACAAAG rs2534805 30541820 rs2516650
30550400 ACGTTGGATGTAGGTATACCTGTGCCATGG
ACGTTGGATGGGAAGGGATAGCATTAGGAG rs1362119 30555226
ACGTTGGATGCAGTGCCTTGATACCTGAAC ACGTTGGATGACAGCCTGGATGGCTTATAG
rs1468079 30562131 ACGTTGGATGAGACATCATGACCATTCACC
ACGTTGGATGAGGCATTCAAATTGGAGAAG rs2524222 30566921
ACGTTGGATGCCAAGTGGTAAGTGAGATAG ACGTTGGATGTCTCCCAGAACTTATCACAC
rs2516640 30572208 ACGTTGGATGCCCAACCCTGTAAAATCCAG
ACGTTGGATGTTCTTAGCCACAGTCAGCTG rs2074505 30576689
ACGTTGGATGCAAGTTGCCCTCTCTCATTG ACGTTGGATGTTTCACCTCTTTTCCTCGGG
rs3130242 30593090 ACGTTGGATGTCACCTTTCCCACAACTCTG
ACGTTGGATGACAAACAGGAAGGAGGCAAG rs1264444 30603213
ACGTTGGATGATGTTGACCAGGATGGTCTC ACGTTGGATGTAATCCCAGCACTTTGGGAG
rs1264440 30606834 ACGTTGGATGAATTCTCCCTTTGGGACAGG
ACGTTGGATGGGGATTATGCTGGAGGTAGC rs1264437 30609619
ACGTTGGATGACGCTGGCCTACATTTCAAG ACGTTGGATGTTTTCCTGGAGAGGAAGAGG
rs1264424 30624639 ACGTTGGATGGAACTCTGACACAGGATCAG
ACGTTGGATGCCACCCCATGAGGAATAATG rs1264423 30627013
ACGTTGGATGCGGTGCATCTTTCATATGAG ACGTTGGATGCCATGGAACACTCCTGAAAG
rs2252745 30635057 ACGTTGGATGTTGGGAGGACAAAAAGGCTG
ACGTTGGATGTGTGCCGGAAAAAACACAGG rs3132607 30645288
ACGTTGGATGAGGAGTTTGAGACCAGCCTG ACGTTGGATGTGAGAAGCTGGGACTACAGG
rs2394388 30654985 ACGTTGGATGGAGGAGTATGGTAGGAGATG
ACGTTGGATGAAGCCAGTCTTTGCAGTAGC rs3132608 30665408
ACGTTGGATGGCACCCACTGACAGTAAGAG ACGTTGGATGTAGGGAGAAAGATCGAAGGG
rs1127955 30676469 ACGTTGGATGAAACAGAACCTGACACCAGC
ACGTTGGATGTCCCAAATGTTCCCACAAGC rs1124795 30677163
ACGTTGGATGTCCTGACCCCTATCATCCTG ACGTTGGATGTATGCTCTGGGAGCCCTCAAC
rs1076829 30682989 ACGTTGGATGCAGGCACACAGCTTTTTCAC
ACGTTGGATGTCAGTTGGAGAAACCCACAC rs1076828 30684025
ACGTTGGATGTGATCTGCAACCTATCCCAG ACGTTGGATGTATGGCTAACTTGTCCTGGC
rs2285320 30696449 ACGTTGGATGATGGCGACTCACGCTCCCTG
ACGTTGGATGTAGAGGTCCCAAGGTAGCTG rs2394392 30705580
ACGTTGGATGAGAGTTCCTCTGACCCAGAC ACGTTGGATGTTGCAGCAGAGCTGGGACAAG
rs2239888 30705674 ACGTTGGATGCCTCTGTACTTTATTTTCTAC
ACGTTGGATGTGAGGAGACAGGCAGGGTAG rs1075496 30714001
ACGTTGGATGCCATGCTTTTTGCAACTGCC ACGTTGGATGTTCCATCCCTAGTTTCTGCC
rs3130644 30716427 ACGTTGGATGAAGTGCTGGGATTACAGCTG
ACGTTGGATGCAGACAGCAGGTATGGTAAG rs3094090 30725716
ACGTTGGATGACCTGTAGTCCCAGCTACTC ACGTTGGATGTCTCGGCTCACTACAATCTC
rs2239886 30726450 ACGTTGGATGGCTCTCTCTAAATGCTAGGC
ACGTTGGATGAGCAGTCAGCATCAAAGCTC rs2394394 30733626
ACGTTGGATGACCTGAGATCGGGAGCTTGA ACGTTGGATGTTACAGGCATGCACCACCAC
rs2075015 30736108 ACGTTGGATGAGCTTGGCTTTTCTCCAGAG
ACGTTGGATGTCCATGGAGTAGGTACAAGG rs25525 30746293
ACGTTGGATGATCCCCTTTGGGTGAATCTG ACGTTGGATGAGACTTGTCATTCCAGGTCC
rs2244011 30750829 ACGTTGGATGCAGACTGTTTGAGCCTGTTG
ACGTTGGATGAAGTTGAAAACCTCCAGCCC rs1059612 30764464
ACGTTGGATGCCCCCCTCATTTTGACATCC ACGTTGGATGTCATGGCCCACATGACTGTG
rs3129973 30776893 ACGTTGGATGAGTTCCCAACCCAAATCCAG
ACGTTGGATGGATGCACAACATCAAGAAGC rs2894045 30788522
ACGTTGGATGGGGCACCTTGAAAAAAGAGC ACGTTGGATGAAATATGGCTCTGTTCCGCC
rs2394402 30789242 ACGTTGGATGTTTCTGCAACCTCTGCCTCC
ACGTTGGATGTTTGTGGCATGCGCCTGTAG rs3130673 30802269
ACGTTGGATGTCTTTAAGTGGATGGGCTCG ACGTTGGATGTGGCAGGCAGAGCAATTTAG
rs3131041 30810816 ACGTTGGATGAGGTTGAAGCGATTCTCCTG
ACGTTGGATGACAAAAGTTAGCTGGGCGTG rs1264377 30820115
ACGTTGGATGAAGACCACTTCAGAGTCCAG ACGTTGGATGGGAGAGGTGGTCATGATCAG
rs2394403 30823632 ACGTTGGATGCTATTCCAAAACATCACTGGC
ACGTTGGATGCGGCCTATTTCTAGTCTTTTG rs1264364 30831067
ACGTTGGATGAGCCTCCCACCCACTCAAAG ACGTTGGATGTTGGGTGGTCGATGGGACTG
rs2894046 30837877 ACGTTGGATGCCATGGTTGAAGGAGAAGAG
ACGTTGGATGATCTTCTGTGGCAGACGTAG rs1264352 30845173
ACGTTGGATGCTTGGTACAAGTGAAACTGG ACGTTGGATGGCTCTTGCTCTTTCTTCTGG
rs915664 30850194 ACGTTGGATGTATGACAGCACGTTTCTGCC
ACGTTGGATGCCTCAAGGAGGCAGTTAAAC rs2535338 30860692
ACGTTGGATGGCCTGGCAACATAGCAAGAC ACGTTGGATGTCAGCCTCATGAGTAGCTGG
rs2535335 30868020 ACGTTGGATGACCCCTCATCTCCTAAGCTC
ACGTTGGATGTGAGCTGTCTTCCTTGCCTC rs2250264 30876536
ACGTTGGATGAGGAGGGAAGGAAGTATAAC ACGTTGGATGGAAACTGTCACCACAATCAAG
rs3095354 30891957 ACGTTGGATGGCTGCATAATAAATTGCCCC
ACGTTGGATGGTGTGTATGTGTTTAAGAGAG rs1264332 30901222
ACGTTGGATGGGAAAGAGATTCAGGCTTGG ACGTTGGATGCCTTTCTGACCTCTCTCTTG
rs2855542 30912003 ACGTTGGATGGAAACTAGGGCAGAGATCAG
ACGTTGGATGTCTAAGCCGTTGTTTATGGG rs3130799 30921946
ACGTTGGATGTGTGACTGATGGAGACCAGG ACGTTGGATGTGCATCCTCATGGTGAGCAG
rs1264314 30926804 ACGTTGGATGCTCCAAAAGAGGTGTGCCTA
ACGTTGGATGCCAGACTGGGCAACAAAATG rs1264297 30940639
ACGTTGGATGTCTAAGAGCCACTTCTCAGC ACGTTGGATGTGTTTAGGGATCTGTGTGGG
rs2532936 30950044 ACGTTGGATGAAAGAGCCTGCAAAAGCCAG
ACGTTGGATGTAGTCATGGGTAGGGTATGG rs3132571 30961148
ACGTTGGATGTTGCCTAGAGCTGAGTTGAG ACGTTGGATGTCAGTGGCCGAGAAAAACAC
rs2240804 30976671 ACGTTGGATGAAAGGGGCAGAGCATGGAAG
ACGTTGGATGATCTTGGCATGGGCCAGATC rs2530715 30989357
ACGTTGGATGTTGGAGGTTGTTGTGGGCAC ACGTTGGATGGGCCTTTGAGGCCACATCAA
rs2517441 30998212 ACGTTGGATGCAAGACTGCATACAGGAATAC
ACGTTGGATGCCATCCTGGTCTTAATCTTC rs2517434 30999873
ACGTTGGATGATCACCGGAAAGACCAAAGC ACGTTGGATGGATTAAACCATGGCCACTGG
rs2523927 31009436 ACGTTGGATGAAACTTGGGCCAGTGTCAAC
ACGTTGGATGATCGAGCCATTGCACTCCAG rs2253417 31025801
ACGTTGGATGAAACCTTCCCCCAAAGACTG ACGTTGGATGCAACATGGCAGATTAGCATC
rs1619376 31039164 ACGTTGGATGAAGAGAAAAATGGGCCCAGC
ACGTTGGATGTGAGTCAAATGTGAGGGTGG rs1632866 31047876
ACGTTGGATGGGGTTCTTTGTGTTATACTTG ACGTTGGATGCCCACTGGAATAACATACTC
rs2523897 31049548 ACGTTGGATGCAACTGCAGACTCCAAGGTG
ACGTTGGATGTGGTGTTAGAGCCTGCAGC rs2844670 31061565
ACGTTGGATGTGCCTCTTACTTGTGCCTTG ACGTTGGATGCACCTCCTTGAATGGAAGTG
rs2252195 31075647 ACGTTGGATGAACTTCTTAGCTTCTATAAT
ACGTTGGATGCTTTGTTTTAGAATTTTTAAAAC rs2517523 31082273
ACGTTGGATGGAGAGGTCACTAGCATTAGC ACGTTGGATGGCCTTTTGAGCCATCTCTTG
rs2523882 31097806 ACGTTGGATGAAGACAGAGGTGAGGAATGC
ACGTTGGATGTAAAACACAGCCTCCTTGGG rs3130959 31112196
ACGTTGGATGGGCCAAATTGACTTTTCACC ACGTTGGATGAATCTGGTTTGCCAGCACAG
rs2517407 31122306 ACGTTGGATGCCATGTTCAACCTTTGGAGG
ACGTTGGATGGCTGTTGGACAGTGAAATGG rs1064190 31130758
ACGTTGGATGAATGCAGTGCGTTGTCCCAG ACGTTGGATGAACTACAGCCTCTGCACCAG
rs3132549 31142686 ACGTTGGATGTTTCACCATCTTGGCCAGGC
ACGTTGGATGCTTGTGCCTGTAATCCCAAC rs1265103 31156625
ACGTTGGATGGGCACAAAAATGGTAAAGGG ACGTTGGATGTCATGTCTGTCTTCCCTTCC
rs1265099 31161025 ACGTTGGATGTCTACTGATAGTTCCTGCCG
ACGTTGGATGTAAGCCTACTCTCCTACCTC rs1265114 31173035
ACGTTGGATGATCCTACCTGAGGCTGACTC ACGTTGGATGCTGGGTGACAAAGCGAGATC
rs1265112 31173867 ACGTTGGATGTCTGAAGGTTGAACCTGAGG
ACGTTGGATGACAAAGATGCCACCTCCTTC rs130078 31174413
ACGTTGGATGAGTTCCCATGTCTGGCTGTG ACGTTGGATGGGTGACCCTGGTTGAGAATC
rs2240059 31176474 ACGTTGGATGGTTCTGAAGTGGCCAAAGCC
ACGTTGGATGGCACTGAGTGTGCTGCAGAG rs130075 31178112
ACGTTGGATGTGATCGTTCGGCAGCTGCAAG ACGTTGGATGTCATCTTCTGCTGCAGCGAG
rs130076 31178340 ACGTTGGATGATCGTTCGGCAGCTGCAAGAG
ACGTTGGATGTCATCTTCTGCTGCAGCGAG rs130065 31178358
ACGTTGGATGATCGTTCGGCAGCTGCAAGAG ACGTTGGATGTCATCTTCTGCTGCAGCGAG
rs2073716 31178855 ACGTTGGATGAGGTTGGAAGAACACACAGG
ACGTTGGATGCCATTCCTCCCTCAAACTTC
rs720466 31181582 ACGTTGGATGTGAAGCCTCGGGTATCTAGG
ACGTTGGATGATTCTGGTCCTGACCCTCAC rs720465 31181654
ACGTTGGATGTCTCTCAATAGCCTGCCCTC ACGTTGGATGTAGAGCTCACGGGCTAACTG
rs1265162 31193347 ACGTTGGATGCCCAAACAGGAGATCCTATC
ACGTTGGATGCCTGAGGGTAAAAACAGTGC rs915660 31199448
ACGTTGGATGGTCTTGGAGAATGAGTGAGG ACGTTGGATGTCCTACCTCCTCCCAAAATG
rs885701 31199563 ACGTTGGATGTCTTCTCTGTCAACCACATC
ACGTTGGATGAGTGCATGCTGGGTACATGG rs1052989 31202267
ACGTTGGATGGGAGGCACTAAATATTCACG ACGTTGGATGTTGAAACCTCCTGCATCCTG
rs1265181 31211656 ACGTTGGATGTTTGGCCTAGTTTGAGTGCC
ACGTTGGATGGCTGCACAAACAACTTTCGC rs886389 31222612
ACGTTGGATGAGAAAGAAAGAAGAGAGAGAG ACGTTGGATGGTCCATTGAATGGAGTATAGC
rs1793899 31225739 ACGTTGGATGACCTCTCTGCTCTCTGTCTC
ACGTTGGATGTCCTTGTCAGGGACCACAAG rs3132502 31239363
ACGTTGGATGCAAGACTCCTTTCCTGTAAC ACGTTGGATGATCGTGCCATTGCACTCTAG
rs1793895 31247789 ACGTTGGATGTCTGAACCCACACAGTACAC
ACGTTGGATGTGGCACAGTCAGAATAAGGC rs1793894 31252511
ACGTTGGATGTTTCTCCATGTTGGTCAGGC ACGTTGGATGAATCTCAGCACCTTGAGAGG
rs3134756 31269208 ACGTTGGATGAAAACATTGCAGGAGCTGAC
ACGTTGGATGCAGCTTTATCAGGTTGGTTTC rs1793893 31272501
ACGTTGGATGTACCATGAATATAGCTATCG ACGTTGGATGTTTGCCTGAAGGACTGAAAC
rs2394948 31275364 ACGTTGGATGGGGTCTAGAGAAGTAGGTTG
ACGTTGGATGGGCAATACAGCTGCATTCAG rs1793892 31275440
ACGTTGGATGTTTGCATCCCTAGTCCTGAG ACGTTGGATGTACAATCCTTCCCAAGGTGG
rs3130542 31286439 ACGTTGGATGGTCTGCTAAACACAGGTTTC
ACGTTGGATGTTATGTGACCCCCTCAAAGG rs2040748 31297875
ACGTTGGATGAGCAATCACAGCAAAGGAAC ACGTTGGATGTCAGGAACACTGAGAGAATG
rs2253288 31301099 ACGTTGGATGCAAAGCCACAATGAGATACC
ACGTTGGATGAGCCTCACCAGCATCTATTG rs2253487 31303455
ACGTTGGATGTCATGCTGAAAGGCTGTGTG ACGTTGGATGAGGTCAATCTTCTCCAGAGC
rs2853941 31303557 ACGTTGGATGGTGGTCCCATGAATGCTTTC
ACGTTGGATGAAGTTCATTGACACCCCCTC rs2844604 31304836
ACGTTGGATGCTGAAAGTGGACTGTGAAATG ACGTTGGATGTGAGACTCAAGACTGGCTAG
rs2853939 31304971 ACGTTGGATGAAACCCTAGCCAGTCTTGAG
ACGTTGGATGTAACTCCTCTTTCTGGGCAG rs2524059 31305152
ACGTTGGATGCAGTGACTTTGTTGCCTTGC ACGTTGGATGTTCTCCAAGTGTGGACACAG
rs2844603 31305183 ACGTTGGATGATTCCACTTTACCCAGTGTC
ACGTTGGATGTCAAGGTTTCTTTCTCCAAG rs2853938 31305806
ACGTTGGATGCCTGGAGGATGAGCAATGAC ACGTTGGATGTTGCAGTGCTCCTGCTCCCA
rs2524058 31305898 ACGTTGGATGTGGGAGCAGGAGCACTGCAA
ACGTTGGATGAGAAATCCCAAGGAGAGGCC rs2524053 31306798
ACGTTGGATGGACTTTTACGATCATCACTTC ACGTTGGATGTTTCAAGGAAGAATCTATAG
rs2853935 31308207 ACGTTGGATGCTATAATCAAAGCCTGGGAC
ACGTTGGATGGGAAATGCAAGAATGAGAGC rs2853933 31308417
ACGTTGGATGTTCCCTCATGTTGTTGCTGG ACGTTGGATGACAGCTACGGGTCTATCAAG
rs2524151 31316283 ACGTTGGATGCCTTCAGATAAGGTATTGGG
ACGTTGGATGTTGGATCAGCAGCTCTTTTG rs2524123 31319639
ACGTTGGATGTCCCCAAGAGGTTTTCACAG ACGTTGGATGCTGCAGTGGTAGAAGAGAAG
rs2247056 31319815 ACGTTGGATGTGCATGGCTGTAAATTAGGC
ACGTTGGATGAGGGCTGTCTAATCATTCCC rs2524089 31320847
ACGTTGGATGCCCCTTCCTTGTATAGTTCC ACGTTGGATGTACAGGTCTGTCCCACCATC
rs364415 31327348 ACGTTGGATGTTGAACCATGAGGAGGAGTC
ACGTTGGATGTCTCCTCTCACACCATCCAG rs3130690 31340260
ACGTTGGATGATGAGGTCATGTGAGTGTGC ACGTTGGATGTTCCTCCGTATCTGTCTGTG
rs2524227 31356125 ACGTTGGATGAAAGAGAATGCCCTGAATGG
ACGTTGGATGAAAAAGAGTAGAGCCCCTGG rs2854008 31366306
ACGTTGGATGAAGACCCATTTGCTGCTTCC ACGTTGGATGTGGGAGGGCCTTGAAAATAC
rs709052 31376822 ACGTTGGATGAGATCACACTGACCTGGCAG
ACGTTGGATGTTCTATCTCCTGCTGGTCTG rs2250295 31384198
ACGTTGGATGGAAAACAAATCCTAGCCAGTC ACGTTGGATGCGATAGTTCTGAAATCGTAGG
rs2596548 31384333 ACGTTGGATGAAATATGGTGTCCCTGGGAC
ACGTTGGATGGAGTGGAAGAGCAAGACAAC rs2853996 31386696
ACGTTGGATGCCATCATCCCTCACTTGAAC ACGTTGGATGGCCACCCCAGATCTTTATTC
rs2596438 31393338 ACGTTGGATGAAGTATGACTCATTCACAGG
ACGTTGGATGGTCCATTGTTCTTCAGGAAC rs2853976 31399027
ACGTTGGATGTACTTCTGATCCCCTAGGAC ACGTTGGATGAGCAGCCTTCCATAGACATC
rs2244020 31401021 ACGTTGGATGATGAACAGGACCTTCCACCC
ACGTTGGATGAGCCACCACACCTTCTTCTG rs2523466 31416809
ACGTTGGATGTATAACTGTCCCAGCTCCTG ACGTTGGATGTAGGAAACATCCCCACCTAG
rs2523454 31421660 ACGTTGGATGTACTCACCCGGATCAGAATC
ACGTTGGATGATGAAAATGCAGACCCGCAG rs2301749 31425152
ACGTTGGATGTTCATTGGATGAGCGGTCGG ACGTTGGATGTCTCAGCGGCTCAAGCAGTG
rs2301747 31425383 ACGTTGGATGTGAAGTGTGGCGGTAACGGG
ACGTTGGATGTGCTGGTGAGTGGCGTTCCT rs2256184 31434180
ACGTTGGATGTCTCTTGAACTCACTAGGGC ACGTTGGATGACTATTTGCTCCCTCTGAGG
rs2848716 31441607 ACGTTGGATGTGAAACCCCAATGTCTCACC
ACGTTGGATGTGAGCCCAGAGTTGACAGAG rs2516446 31446421
ACGTTGGATGTCAAGTGATCCTGCTCTCTC ACGTTGGATGTAGTAAAGAGGGCAGGCATG
rs2516470 31460915 ACGTTGGATGAGTTAAGAGATTCCCTGACC
ACGTTGGATGAAAGACAGCACATTCTGCCG rs3099847 31476996
ACGTTGGATGAGGGGCTCCTCACTTCCCAG ACGTTGGATGTCAGCTCCCCGCCCAGCCA
rs2596552 31477071 ACGTTGGATGAGGCAGAGGGGCTCCTCAC
ACGTTGGATGTGAGGAGCGTCTCCGCCCG rs2596472 31482726
ACGTTGGATGTTTACCAGATGTCTGAAAGG ACGTTGGATGTATCAATTCGCCCATTGCAG
rs2523674 31490746 ACGTTGGATGCAGAAAAGACTGGGAAAGCC
ACGTTGGATGTTGCAGTGAGCTGAGATTGC rs2904786 31510355
ACGTTGGATGGTTGATGGCACCTTCAGAAG ACGTTGGATGAAACCCAAAGATGGGTCAGG
rs2855804 31521290 ACGTTGGATGTTCTGGTGCTGCCTTTTGTC
ACGTTGGATGAACTGCCATTAGCATCAGGG rs3132464 31531399
ACGTTGGATGTTTCTCTCTTCAGTTGCCCC ACGTTGGATGGGGAGGAAGAAAAAAGTGGG
rs2516400 31553393 ACGTTGGATGAGGTGGACAAATCACAGGTG
ACGTTGGATGTCAACGGTGTTTCTTGGAGG rs3130638 31560032
ACGTTGGATGTGAGGTCAGGAGTTCAAGAC ACGTTGGATGCCATGCCTGGCTAATATTTG
rs11796 31564553 ACGTTGGATGTTTTGACTGTCCATTGCAGC
ACGTTGGATGCGTGTGCATTAGCAAAGTGG rs2239709 31570511
ACGTTGGATGTAGAGATGACTGGCTTCTGG ACGTTGGATGTTGCTATACTTCGGGTCACG
rs2857607 31580348 ACGTTGGATGACTTTGAGAGGCTGAGGTTG
ACGTTGGATGTTTCGCCATGTTGGACAAGC rs2230365 31588298
ACGTTGGATGTACACCGATTTCTTCCTCCC ACGTTGGATGGGGTCTCCCCATCCTTATTC
rs2844490 31595707 ACGTTGGATGGCCTTTTGCATTTGCCATGC
ACGTTGGATGGTGGAGAAAGACTGAGCTAG rs736160 31601908 rs361525 31606263
ACGTTGGATGATCAAGGATACCCCTCACAC ACGTTGGATGACACAAATCAGTCAGTGGCC
rs986475 31619673 ACGTTGGATGGTCCCTGAACACTGTCATTC
ACGTTGGATGAAACACATGGCTCACCCTTC rs2857595 31631860
ACGTTGGATGTTGATAAGACTTGGCCAGAG ACGTTGGATGTGATCTCATCTTTCCCCCAC
rs2051552 31643098 ACGTTGGATGGCCAACATAGTAAAACCCCG
ACGTTGGATGAAGTGATTCTCCTGCCTCAG rs2844476 31645038
ACGTTGGATGATTGCACCATTGCACTCCCG ACGTTGGATGAGATGCTGGAGTGGCCTCTG
rs2736181 31646906 ACGTTGGATGTCTCTCAGCATCCCCTCTAG
ACGTTGGATGAGACAACGTGGAAGGAGGAG rs2736160 31662291
ACGTTGGATGATAACTGGCCAGATAGGGTG ACGTTGGATGCTTTTCCCACCTAGTTCTGG
rs750332 31670185 ACGTTGGATGTAAGCAGGTTGGAGAAACGC
ACGTTGGATGTGTTAGCTTCTGAGGGATGG rs1052486 31673871
ACGTTGGATGACAGTGATGGTGGGAGAAGC ACGTTGGATGTCAGTTCTCTCAGCTTCTGG
rs3117583 31682962 ACGTTGGATGCCGACAGGTCTCTAAAGAAG
ACGTTGGATGAGTCTTTCGGGTACACTCTG rs2894225 31692409 rs928814 31695049
ACGTTGGATGGGTTCCAGCAGTCTCCTAAG ACGTTGGATGAGATGACTCACCGGATACTG
rs805256 31698902 ACGTTGGATGTCCAGGTCCAAGATCATGTC
ACGTTGGATGTACTGGACTCAATGAGCAGG rs805290 31711788
ACGTTGGATGGAGACTTTGTGCAGGGTTGT ACGTTGGATGGGGAATGAGAAAAGGAACTG
rs805281 31724677 ACGTTGGATGCCTCTTCCAAGCTAAGAACC
ACGTTGGATGAAAGCACTAGCACCTTCAGC rs805289 31740426
ACGTTGGATGAGTAGCTGGACTACAGGTGC ACGTTGGATGACAGAGAGAGACTCTGTCTC
rs376510 31751388 ACGTTGGATGGTGGAGTGACGGAAGATATG
ACGTTGGATGAGGTAAGGGTAGAGCTGTTG rs805292 31753147
ACGTTGGATGTTAATCTCCATTCAGCCCCC ACGTTGGATGAGAAGCCATCAGTGAGTCAC
rs3131382 31771118 ACGTTGGATGTCGGATCTCTAGGCTGGATC
ACGTTGGATGACGAGCCTGCAAAAGGAGCG rs1150793 31789133
ACGTTGGATGAATCCTTCCCCTACCTCACC ACGTTGGATGTTACCTGGAGATGACCTCAG
rs707935 31806157 ACGTTGGATGTACATTTATTCCCTGAGCCC
ACGTTGGATGGTTATGCATATGCACAGATG rs707932 31810029
ACGTTGGATGAGGAGAATCACTTGAACCCG ACGTTGGATGCCTGCACTGACAAGTATGAC
rs707928 31814030 ACGTTGGATGCCTGTGCTGTGTTTTCCAGC
ACGTTGGATGAAAACCTAGGATCATGGGCC rs1150749 31830029
ACGTTGGATGAATCGCTTGAACCTAGGAGG ACGTTGGATGACTCTTTTTGCTCAGGCTGG
rs480092 31836340 ACGTTGGATGTACCATACCTGCAACTGGAG
ACGTTGGATGTGGTGAAGTCTGGTAGCATG rs2075799 31850226
ACGTTGGATGTTATCAGGGCAGTCATCACG ACGTTGGATGAGTCTGAGAAGGTACAGGAC
rs539689 31857089 ACGTTGGATGATCCACCTCCTCAATGGTAG
ACGTTGGATGTGTGTAACCCCATCATCAGC rs2763979 31866094
ACGTTGGATGATCTTACTCGGGACTGTGAG ACGTTGGATGCACCTCCTTCCTACTTTCTC
rs3130679 31879245 ACGTTGGATGCATTGTTCTGAGACCAAACC
ACGTTGGATGGGGCACTTCAAGTAGATAGC rs574914 31890829
ACGTTGGATGGCCAAGATGGAAGTTAAGCC ACGTTGGATGGGAGAGTCTGTAAGAAGCAG
rs2021007 31890874 ACGTTGGATGGGAAGTTAAGCCTTGGAGAC
ACGTTGGATGGTGATTGGAAAGGAGGTCTC rs660550 31909117
ACGTTGGATGGTGACACCAAGGCACTCTAC ACGTTGGATGACTGCTCTGTATCCTCTGCC
rs605203 31918651 ACGTTGGATGTTTAATCTTTGGCGGGAGCG
ACGTTGGATGACCTGGCATGCTCTGATAAG rs589428 31919809
ACGTTGGATGATGGGATCTGAGCCCCTTGT ACGTTGGATGTGTCCAGGCATGGAGTAGTG
rs612496 31931785 ACGTTGGATGCAATATCACGATCTGGGCTC
ACGTTGGATGAGGCTGAGGCAGGAGAATTG rs558702 31941915
ACGTTGGATGTGTGAGCACACTCAGCAGAG ACGTTGGATGTGCATCCGTGGCACCTCTCA
rs2763982 31944190 ACGTTGGATGTCCAGTGAGAGCAGAAATAC
ACGTTGGATGTGTCCCATCTACATTCCTAG rs3020644 31956403
ACGTTGGATGCACTTCAGAGAGGTTTCATG ACGTTGGATGTGACCCACAGAAGTCTTTTC
rs1265911 31962348 ACGTTGGATGAGTGCTCAATGATGCCCAGG
ACGTTGGATGAATCTCGGCACTTTGGGAGG rs2854340 31963783
ACGTTGGATGTGTCCCAACAGTGCTTGTGG ACGTTGGATGCCTCCAAGAAGTCTTCTCAG
rs609061 31971951 ACGTTGGATGCCTGTTTATTCCCTGTAATGG
ACGTTGGATGTGATTACAGGTGTGAGCCAC rs1270942 31980650
ACGTTGGATGTAGCTCTAGAAGGGCTTAGG ACGTTGGATGATAGACTGCGTCACTTCAGC
rs419788 31990590 ACGTTGGATGCCTTTCTTGAAACCAGGTGG
ACGTTGGATGTTGTTCACCAGTGTGCAGTG rs429608 31992054
ACGTTGGATGACTGTACAGCATGGAGCTGG ACGTTGGATGAGAGTGTGCTGTCTGAGGAG
rs416002 32000424 ACGTTGGATGACATGTGTGCAGGTGAGTTG
ACGTTGGATGTGTCTGCACAAGGAGAGAAG rs2746392 32009805
ACGTTGGATGTCTGTTGGTGCAGTTGCTTG ACGTTGGATGGGAAAAGAAAAAGTGGAGGG
rs2734323 32020483 ACGTTGGATGAGCTCACTGTCTTGTGGGAG
ACGTTGGATGCAAGAAGAGAGGGACAGGAG rs3130677 32034947 rs433061 32043622
ACGTTGGATGTTCCCAAACCCTCACTGTGG ACGTTGGATGGGGAGAAGACAGGGGATTAG
rs916139 32046588 ACGTTGGATGTCACCATCTCAGGCCTGGAG
ACGTTGGATGCGTGGAAGCCGTACAGGTTC rs3117189 32078519
ACGTTGGATGCCCAGGCTGATATCAAACTC ACGTTGGATGATCCCAGCACTTTGTAAGGC
rs204879 32087748 ACGTTGGATGAGAGCAAATGCAGAGACTGG
ACGTTGGATGCTCCAACTCCACCACAAAAC rs2021783 32089404
ACGTTGGATGGAAGGAACTCAGTTTGTCAG ACGTTGGATGTGTCAGGCACTGACCAAGTC
rs2239688 32098814 ACGTTGGATGAAGGCTTCCATGACCTCCAG
ACGTTGGATGAATGGAAGCCACCTGACCAC rs204896 32108695
ACGTTGGATGACAGTCCTCACCGGTGAAGC ACGTTGGATGATGTGTTGGCCGGGGTACAC
rs393544 32118867 ACGTTGGATGTCTCTTACCCAAGGCTACAC
ACGTTGGATGATACAGAGAGCTGCCCTTTC rs1269852 32119789
ACGTTGGATGTCCAAACCCTTCCTTCTGAG ACGTTGGATGCAGGATGAAAGATGGGAGAG
rs204894 32134359 ACGTTGGATGGGAGATGACCCAACATCCTC
ACGTTGGATGCTGAGGAATCATCAGAGGTG rs421602 32135422
ACGTTGGATGTCTCCTTTACAGCTTGGTGC ACGTTGGATGGTAACAGAGGCAGCTGTTTG
rs2071291 32140276 ACGTTGGATGGCACGTAAGCAGTGCAAGGC
ACGTTGGATGTTCGTGGTGCCCACGCACG rs2269425 32150480
ACGTTGGATGGGGAACAGAGGTTTATGGTC ACGTTGGATGACAGCCACTTCAAGTAGTCC
rs1269839 32163718 ACGTTGGATGAAACCCTCTTCCTTGTCTCC
ACGTTGGATGGCTGTATCCACATTCACTTC rs408359 32168920
ACGTTGGATGCACTGATCTTGACAACACAC ACGTTGGATGAAGCTGCTTACAGCCTAAGG
rs204996 32176422 ACGTTGGATGTCTGGCCTTATCCCTAACAG
ACGTTGGATGGCTCTCTTGGCAGAATTTGG rs204989 32188886
ACGTTGGATGGAAACATGGAGTCATGAGGC ACGTTGGATGGCACCTACTTCATAGGGTTG
rs2071280 32191654 ACGTTGGATGACTGCAGTTTGTCTGCTACG
ACGTTGGATGTAAGGAACTCGAGTTGGCAG rs2071277 32210496
ACGTTGGATGAGGAATGAGCTAGGATGGAG ACGTTGGATGCACTGGCCTGTAATTATGGG
rs2856433 32221133 ACGTTGGATGCTCTCGTAGAGCTTTCATTC
ACGTTGGATGACCTGCTCATTTTCTCCAAC rs375244 32230293
ACGTTGGATGAAATAGAGACGGCCTCCAGG ACGTTGGATGTGACGAGGTTCTTCCTGGAG
rs2849015 32237569 ACGTTGGATGAAACTGCTCATCACCACACC
ACGTTGGATGATGCCCCAATCATCTCTCAC rs3130316 32250273
ACGTTGGATGCTTCCTTTCCAATCTTTTGG ACGTTGGATGCAAGGAGACTACTATCATCAC
rs1150763 32253575 ACGTTGGATGTGGGACTATGTGAAAAGACC
ACGTTGGATGTTGATTCCATTCTCCCCGTC rs3130338 32270791
ACGTTGGATGAGAGAAGCAGGAGTAAGGTG ACGTTGGATGCAGCATCACTAAGGATCAGG
rs1265788 32281025 ACGTTGGATGAGTCAGGAAACAACAGATGC
ACGTTGGATGTTACACTCCCATCAACAGTG rs926070 32283275
ACGTTGGATGGCCTTTAGAAAATGGTCCAC ACGTTGGATGTCCCACTAGGTCTTTGAAGG
rs2038191 32290024 rs491870 32298352 ACGTTGGATGAAAGAATTGGGACTTGCCTC
ACGTTGGATGTGGTGTATTTAGACCTACAC rs1018433 32308410
ACGTTGGATGTGTCCTAACTTCCTGGGTAC ACGTTGGATGTGTGCTACCCATGCAGTGTG
rs513095 32314754 ACGTTGGATGATCACTCACCACTCACATGG
ACGTTGGATGGCCCCCAAGGAATAAGAAAC rs742697 32318423
ACGTTGGATGAGGGAAAACTTTCCCTTTGG ACGTTGGATGGGGTGCATACTACTTTAACC
rs523627 32318719 ACGTTGGATGTGACCTGCTGATAAACTTTC
ACGTTGGATGTGACACATCATTCTCTCACC rs2077333 32320455
ACGTTGGATGACCACCACCTAAGTTTCCAG ACGTTGGATGAAGCAAGAGATGGCTAGTGC
rs2395143 32320463 ACGTTGGATGACCACCAAAGTTTCCAGAAC
ACGTTGGATGCTGTCAAAGCAAGAGATGGC rs504703 32320902
ACGTTGGATGAGAAGTGACAGGGAAGCTAC ACGTTGGATGTTCTTTGTACCAGCTAGGCC
rs3129958 32327056 ACGTTGGATGACTGATGGTAGGGAAAGGTG
ACGTTGGATGAGAACAGTCCCTTGAGAAAG rs3129960 32327712
ACGTTGGATGATCATGCCACTGCATTCCAG ACGTTGGATGTTTCTAGCTCTGATCGCCTG
rs2022537 32328814 ACGTTGGATGCTTGGATAGGTGATCACTTC
ACGTTGGATGAGGGAAATGAGTATGTTGAG rs2022534 32333790
ACGTTGGATGCTCTCTTTTACCAGTGTGAG ACGTTGGATGCAGTCACTTAGAGGATCTTG
rs2143468 32335906 ACGTTGGATGTATCCACAGAGACAATGTCC
ACGTTGGATGGGGCAGTGGAAGGTATTTAC rs2395145 32338774
ACGTTGGATGCTCACCATCTTTTGGAACTG ACGTTGGATGAAACCCTGTCATTGATCGAC
rs2076542 32343684 ACGTTGGATGTACATGGCTAGCACGAAAGG
ACGTTGGATGATCTCTTCCATTGCTGCCAG rs2076541 32343772
ACGTTGGATGGGATAAGAGCAAAAAGTTAG ACGTTGGATGCTGAGGACACAGCTAATATC
rs2076540 32343828 ACGTTGGATGGAGAGCAATTTCCAAACCTG
ACGTTGGATGCTAACTTTTTGCTCTTATCC rs3129907 32350292
ACGTTGGATGGTTATAAGGTAAGTTGAGGTC ACGTTGGATGTGAATTCTCAGTCAGCTGAG
rs3129927 32360382 ACGTTGGATGCCTGCCACAACATAAAAGGC
ACGTTGGATGAAATGGTGCCTCATAGCGTG rs2143462 32361583
ACGTTGGATGTTAGTGGTACTGGTGTGTCC ACGTTGGATGCAGGTTTTGAAACGTGAGAG
rs2073047 32362481 ACGTTGGATGTTGGTGATTGACACAGTCAC
ACGTTGGATGGCAGGAACTAGGAATTGTGC rs2073044 32365548
ACGTTGGATGGTACTGAGTACACCATCTAG ACGTTGGATGCAAGTAGTCAATATGCCCTC
rs2050190 32365638 ACGTTGGATGCCCTATTAATAGGGTGGACC
ACGTTGGATGAGTGTCTGAAATGCCCTGTC rs2076536 32365910
ACGTTGGATGTCCTTGCCTGCTTCCTTTTC ACGTTGGATGTAACTGTGGGTTGTTTCCCC
rs2050189 32366209 ACGTTGGATGGCTTAGGTCTGATCAATCTG
ACGTTGGATGTATGAACTTGGGTGTCAGGG rs2395151 32369884
ACGTTGGATGAAAACGATGCCCCTATCAGC ACGTTGGATGGTACGTCTAACTGCTGTTCG
rs2894252 32371988 ACGTTGGATGGGGAAAGAAAATGTCTATGGC
ACGTTGGATGTAGATGAGAGTGCAACTTCG rs2395156 32374635
ACGTTGGATGTTGCACAGATGCAAAGATTC ACGTTGGATGAAATGTTTGTGCCATCTAAG
rs2395157 32374682 ACGTTGGATGTTTAAAATGTTTGTGCCATC
ACGTTGGATGTTGCACAGATGCAAAGATTC rs1555115 32381050
ACGTTGGATGATCTATTCCAGCCAGGCTAG ACGTTGGATGCCCATCCTGAAAACCTTACC
rs2076534 32388689 ACGTTGGATGTTTGCAGAGGATAGCAGGAG
ACGTTGGATGAGACCAACTCAGACTTACTC rs2076533 32390050
ACGTTGGATGCTAATAACACACTGTGAAAC ACGTTGGATGAGGAAATCTGAGTATCTTAC
rs2076530 32390339 ACGTTGGATGAGGCCAGTTTGGATCTGAAG
ACGTTGGATGATTAAAGTGGCAGGAGCAGG rs2076529 32390478
ACGTTGGATGTCAGTCTGCCCTCGTCAATG ACGTTGGATGGAGAGCAGATGGCAGAGTAC
rs2294880 32394245 ACGTTGGATGACCTGACAGGAAGCAAAGGG
ACGTTGGATGTAAGTCATGGTAACCTCCGG rs2294878 32394318
ACGTTGGATGTAGGAACAACAGGACATGGG ACGTTGGATGTCCTCTGAGTTCTCTGAGAC
rs2076525 32397124 ACGTTGGATGGCACCTCGTATTTTTATCAAG
ACGTTGGATGTGGCTTTCAATACATATTGC rs2076524 32397192
ACGTTGGATGTACGAGGTGCTATGGTGCAG ACGTTGGATGAGGTCAGTGCTCTGCCTCTAG
rs2076523 32397343 ACGTTGGATGTATTGGGAAGACATCCGGG
ACGTTGGATGTGGCTTCCGCATAGAACAGG rs2395158 32401103
ACGTTGGATGGCTGAGTCACCTTTGGAAAG ACGTTGGATGGGCCTCTGAGATGTAGTTAC
rs3135380 32411189 ACGTTGGATGTAAAATTGGGCATGGGAAAC
ACGTTGGATGGAAATCTGCTAGGCTTAAAC rs2395161 32414066
ACGTTGGATGTTTCCCTCCCCACAATCTAC ACGTTGGATGTCACCTGGACCTGATTGATC
rs2395163 32414323 ACGTTGGATGATCGGCAGCTTGGAAACTAC
ACGTTGGATGGGGCTGGATAATGATGGATG rs2395165 32414658
ACGTTGGATGCAGCTTCCATGTGGTGTTTG ACGTTGGATGTTTGTCCCTCTAGCCCTTTG
rs2395166 32414789 ACGTTGGATGCAGTTCCTATGAAGGATGATC
ACGTTGGATGCCATAGAAACCTTGGAAGTC rs2213581 32415060
ACGTTGGATGCAGTATCCCACAGAGAAGTC ACGTTGGATGGGAGCCTCAAATTATCACTC
rs732163 32421456 ACGTTGGATGACCCCTTTCTAATATCTCTC
ACGTTGGATGTCTTCTATATCGGATAATGC rs732162 32421458
ACGTTGGATGACCCCTTTCTAATATCTCTC ACGTTGGATGTCTTCTATATCGGATAATGC
rs1894552 32422010 ACGTTGGATGGCTCTTCAACTTATGATGGG
ACGTTGGATGGCCACATGATCATGAAGGTG rs2105903 32422201
ACGTTGGATGAAACTACAGACACACCTGAC ACGTTGGATGTCACCTTCATGATCATGTGG
rs983561 32430210 ACGTTGGATGTCATATTGGCCACTCCGAAG
ACGTTGGATGTGAGAAGATGAGAGCAACAG rs3129868 32430931
ACGTTGGATGTATTCCAGCAGACCAGCTTC ACGTTGGATGGAGGTGCTGAGGGAATATTG
rs2395173 32431414 ACGTTGGATGTACATCTCTCAGGCTTGCTC
ACGTTGGATGACTTCCACCTCCCAAATCTC rs2395174 32431433
ACGTTGGATGTACATCTCTCAGGCTTGCTC ACGTTGGATGACTTCCACCTCCCAAATCTC
rs2395177 32431631 ACGTTGGATGATCTGCAACATCAGCAGAGG
ACGTTGGATGAGCCCTTAAAACTGTTAGGG rs2239804 32438079
ACGTTGGATGTGTTACTTCTTCCCACACTC ACGTTGGATGGCTTGGAGCATCAAACTCTG
rs2239802 32438402 ACGTTGGATGCTGAAGCTTTGGGATACCAG
ACGTTGGATGAGGAACAGATGTGGCTCTTG rs1051336 32438914
ACGTTGGATGAGTGTGGATATGCCTCTTCG ACGTTGGATGGGAAAAGGCAATAGACAGGG
rs3177928 32438957 ACGTTGGATGGGTAACTATGTGTGTCTTGC
ACGTTGGATGGCAGAAGTTTCTTCAGTGATC rs7194 32439002
ACGTTGGATGCATGGAGGTGATGGTGTTTC ACGTTGGATGTGCTTTCACTGAGGTCAAGG
rs2213586 32439616 ACGTTGGATGTCTGAGATCCATACCTTGGG
ACGTTGGATGTTGGGAGATCTCTACTGAGC rs2213585 32439672
ACGTTGGATGAACCCCAAGGTATGGATCTC ACGTTGGATGTTCCTTCTCCCCACTCTAAC
rs2213584 32439781 ACGTTGGATGAATGGGTTAGGCCAGTCTTC
ACGTTGGATGGAAGGAAGACAGAAGAATCC rs2395182 32439839
ACGTTGGATGGGCCTTACCCATTCTGTTAG ACGTTGGATGTCAGTCAGACTACTCTCTCG
rs2227139 32439981 ACGTTGGATGGACATTAAGATGAGAGGAAGG
ACGTTGGATGTGGTTTATGGCAGGTTCTAG rs1547422 32453362
ACGTTGGATGTGCATAAGCATTTCACTGAG ACGTTGGATGCAAACCTGTACATGTATCCC
rs1548306 32453442 ACGTTGGATGATAATGTGAGGAGGCTAGTC
ACGTTGGATGATTTCAGAGATTTCGGGATC rs2187824 32465527
ACGTTGGATGCTCTAGCCTTCTTTCTGTCC ACGTTGGATGTTCCAGGGAGACAGAATGTG
rs2187823 32465789 ACGTTGGATGCCAGGATCCAAACAGTGATC
ACGTTGGATGAGTACACAGTAGCTGCTGAG rs2187822 32475997
ACGTTGGATGACCAGGCCTTTGATTTTCAG ACGTTGGATGACTACATTTGGGATACTGGG
rs1974460 32480175 ACGTTGGATGAGCAGGCAAGTCTCACATTC
ACGTTGGATGGTACCTTACTCCCTGTGTTG rs2395199 32482024
ACGTTGGATGTCAGTGCAGTCAGCTGCCTC ACGTTGGATGAGCCACTGAGGGAGTAGTGG
rs2894266 32491452 ACGTTGGATGGCAAATCTGTCCTCCAACAC
ACGTTGGATGGGTGTGGGTTTTGGTGTTAG rs2213583 32499701
ACGTTGGATGTCTGTCTCAGCCCACTTTGC ACGTTGGATGGTGGAAGAGGATACATAGGG
rs1987529 32502240 ACGTTGGATGCCAGTTTTTCAGAGGATGCC
ACGTTGGATGCTGGAACTGAAGCTGAGATC rs2395210 32502763
ACGTTGGATGTTCCCCATACAGCAATTCCC ACGTTGGATGATAACCCAGGATCGTCTAGG
rs2071807 32503145 ACGTTGGATGTATATTCCCCCACCCCATAG
ACGTTGGATGCGTTGACAGTGACACTGATG rs2071806 32503501
ACGTTGGATGGCAACTGGTTCAAACCTTTC ACGTTGGATGGCTGTATGAAGGTCCTCTTC
rs2187821 32504679 ACGTTGGATGGCACTTAGTGCAATTCTGAG
ACGTTGGATGTAGGCCTTAGTGTTTCCAGG rs2157337 32504906
ACGTTGGATGAGGCCTATAAGGAATGAGTG ACGTTGGATGCAGAATGGACTTCAAAGTAC
rs981559 32505329 ACGTTGGATGGCACATAGCAATATGGCTAC
ACGTTGGATGGGAACTAGAATTGCTACACAG rs1987947 32505573
ACGTTGGATGTTCCAAAGTAAGTGAGGCAC ACGTTGGATGACAGTGACCTCAAAATTCCC
rs2395211 32506386 ACGTTGGATGTGGTTTGGGAAGTGGGAGTG
ACGTTGGATGAACTGGGCTTCCTCAGCAGG rs1894554 32506604
ACGTTGGATGGGCAAGGATGATGTGTCTGC ACGTTGGATGTTGGGTGTGATCTGCTCCAC
rs2395213 32506777 ACGTTGGATGAGACACCTGCAAGCCTGCAG
ACGTTGGATGTCCATGCAGCAAGATCCAGG rs2097440 32507071
ACGTTGGATGTTCTGCCCAGGAGACTGTCTG ACGTTGGATGTTGCCATGAGCAGCCTAGGTG
rs2097439 32507201 ACGTTGGATGCTGCTGACACGAGTGGGAAC
ACGTTGGATGCTTTTACAGGCCTCAGAGGG rs2006039 32540959
ACGTTGGATGCAGATGATGAGGTAGGATGC ACGTTGGATGTTACTGTGAACATCAGGGCC
rs1540307 32550985 ACGTTGGATGGAGAGAGTCTATTCCCTTAG
ACGTTGGATGTAAACTAGTTCTCCTACTCC rs707784 32566932
ACGTTGGATGCCTCACCTTTCTGATTCCTG ACGTTGGATGACAGAGCAAGATGCTGAGTG
rs2308665 32570455 rs2395217 32575274
ACGTTGGATGATGTTAGCCAGGATGGTCTC ACGTTGGATGTAATCCCTGCACTTTGGGAG
rs1059544 32578551 ACGTTGGATGGGTTCATAGTTCTCCCTGAG
ACGTTGGATGATGCTGGAGAACAGGACAGG rs2647063 32588521
ACGTTGGATGGACAGTAGCACATGTGAGTC ACGTTGGATGTCTAGACACTGGTAACCCTG
rs2858860 32598186 ACGTTGGATGCTGCAGACCTCACTCTATGG
ACGTTGGATGAGGAGCAGAGAAAAGTCCTG rs2105899 32608060
ACGTTGGATGCCAATCTCTGCTCAAGTGTG ACGTTGGATGACTGGGCTTGAACAGTGATG
rs2040410 32620180 ACGTTGGATGTACCTCATTAGGCAGTTGTG
ACGTTGGATGTGTCCTCCTTGGAAAATGAG rs2213287 32622576
ACGTTGGATGTAGAGACCTCCAGGCTATAG ACGTTGGATGAAACCAGAGTCCCAACCTAC
rs1894385 32655597 ACGTTGGATGGCTGCAGACATATCTAGGAG
ACGTTGGATGGCAAAGCTTCATTGAGGAGG rs2395229 32664446
ACGTTGGATGAAAGCGTGTGGGTGTTCTAG ACGTTGGATGTGGTAAGCATCACTGTCTCC
rs1360 32666380 ACGTTGGATGTCTTCTGGTTTGGTGAGTGC
ACGTTGGATGAAGGGTCACTATATCTGCCC rs1064173 32666475
ACGTTGGATGATATTCTCAGGCCACTGCAC ACGTTGGATGAGGAGGTAGAAGATCAACTC
rs1056316 32666490 ACGTTGGATGGGGTTGTACCTTGAAAAGAC
ACGTTGGATGCATGAATGATGCGACAACTG rs1762 32666522 rs2647027 32674779
ACGTTGGATGAGTACTGTCCCTAGTCACTG ACGTTGGATGCAG1TCCTCATGGACATATC
rs2395231 32676429 ACGTTGGATGTTCATAGAGCATGAGGAGCC
ACGTTGGATGACATTTGAGGGCAAATGAGG rs2647015 32677576
ACGTTGGATGAATGAAGATGACAGGCAGAG ACGTTGGATGACTCACAGAAGCCAAAGAAG
rs2157051 32682878 rs2894283 32692730
ACGTTGGATGCTGTTCATCTCTATTGACTTG ACGTTGGATGCCAAAGCATTTAATGGTTTAG
rs2894284 32693263 ACGTTGGATGAGAACCAAACCTTCACTTGG
ACGTTGGATGGTTATGGGTGTTGTTTAGCC rs2858888 32696820
ACGTTGGATGCTTCAGGGCAAAAGACAATG ACGTTGGATGCCCCTTAAGATGGTCTAATAG
rs2859112 32700110 rs2859091 32703147
ACGTTGGATGTGACTTCCTTTTCTCCCAGG ACGTTGGATGAACACATCAGAAGGCACACC
rs2051599 32711686 ACGTTGGATGAGGAATGTTCTCTGGAGCTG
ACGTTGGATGGACCCTTGGGAAATTTCTAC rs2395252 32713659
ACGTTGGATGAAAGCAGAAGGCCCTGCTGAG ACGTTGGATGACATCACTCTACTGGCCCAG
rs2071800 32716486 ACGTTGGATGTGGGCACTGTCTTCATCATC
ACGTTGGATGTCATAAGAGCCCTTGGTGTC rs2395253 32717006
ACGTTGGATGAGCTTTCCTCTCCCCTTCTC ACGTTGGATGCTGGCTTCCATTTCTTTTCC
rs2213572 32722145 ACGTTGGATGGACAACAAAAAAAATACTTCT
ACGTTGGATGTTTCAGTGAGATCCTGGGTTA rs1573649 32728253
ACGTTGGATGGGATCTGCAGAGCCATCTTC ACGTTGGATGTGAGCTGTGTTGACTACCAC
rs1573647 32728610 ACGTTGGATGCCTCACTTAATTTGCCCTAC
ACGTTGGATGGAAGATTGAATGGCTTAGGG rs2857210 32738722
ACGTTGGATGGAAGCCTTCAATGTTACAGG ACGTTGGATGACTCCAGAAGAGTAGAGTGG
rs719654 32749097 ACGTTGGATGGTGACACTAATAACCCAAGG
ACGTTGGATGGTAGTGAACTTCCATGCAGG rs2157080 32758398
ACGTTGGATGAGAGGACACAGTCATCTCAG ACGTTGGATGCGAGTAGGTACTCTCATTGG
rs2621343 32771751 ACGTTGGATGTATTCCACTCCCAACTCCTG
ACGTTGGATGGCGGATTCCTAATTCTGAGG rs2857107 32782267
ACGTTGGATGGAGTGTTAAAGGTAGAAGCC ACGTTGGATGAAGTGTATCCCATTTTTTCC
rs241447 32793449 rs2127673 32809315 ACGTTGGATGAACTGTCGACGTCACACGAC
ACGTTGGATGTCTGAAGCTGCACCTGGAGG rs1871668 32825643
ACGTTGGATGATACTAGTAGGATCTCAGGC ACGTTGGATGGAAACAACTCCAGGCATTTG
rs1383267 32830393 ACGTTGGATGCTCGGTTCTAACCAAGTAGG
ACGTTGGATGATGTTACCTTGGCGAAAGGC rs241415 32839637
ACGTTGGATGTTTCCTCAATAGGTGTAGAC ACGTTGGATGCTATGAACAATTCTACACAC
rs1029295 32853431 ACGTTGGATGGAATTCACAGGCTTTTAGCC
ACGTTGGATGAGGCTTAATGATGAGAGGTG rs241404 32862944
ACGTTGGATGAATGTCATATGCCTCCTCCC ACGTTGGATGTGCAACTATCTGGACACATG
rs2187688 32868648 rs154985 32877112 ACGTTGGATGACCTGTTGGGAAATGTAGGC
ACGTTGGATGCCATGAGTGAGGATTCCAAG rs151722 32892567
ACGTTGGATGCTGGCCTGAGTTTTGATAAG ACGTTGGATGGGCAAGCTACATAATGGAAG
rs151719 32900606 ACGTTGGATGAGGACACATGGGAGATCTAG
ACGTTGGATGTAACCTCCAGTGGATCCATC rs10679 32913451
ACGTTGGATGTCCAAACAGAGGATGCTCAG ACGTTGGATGTCCCAGAGACTTCTTCTACC
rs1431394 32926004 ACGTTGGATGTATGCACTAACCCATCAGCG
ACGTTGGATGCTTCTTTTCTACTGTCCAGG rs206787 32938141
ACGTTGGATGTGAGGCAGGAGGTCAGCAC ACGTTGGATGTCCGGACCGGAACCGCATCT
rs2567267 32948944 ACGTTGGATGGACTTGTTTTTCATGGCGTAG
ACGTTGGATGCTCCAGCCTGGAGTCTTTAAA rs188245 32955171
ACGTTGGATGATCACTGCCTTTGGTGTTGC ACGTTGGATGACTCCCTGGCCAAATGATTG
rs3135332 32969029 ACGTTGGATGATGTTTGATAGCAGACTGGG
ACGTTGGATGCCTCTCTTCTAGCTACTTTG rs419434 32988697
ACGTTGGATGGGCAGTGTGAACTAAGAGTG ACGTTGGATGAGTGTCTCCAACTATGTGGC
rs3128942 32998935 rs663310 33009016 ACGTTGGATGGTCAGCCTCTGTATAAGGAC
ACGTTGGATGTAGGAGAGAGCCAAATCCAG rs377572 33017193
ACGTTGGATGTTTCCCACCTCCACAGTTTG ACGTTGGATGAAAGCTGAGAGAACCCACAG
rs412735 33026279 ACGTTGGATGTCCAGTCAAAGAGTGAACCC
ACGTTGGATGCATATGGAAGGGTGTGCAAG rs2308935 33038580
ACGTTGGATGCTCCTCTTTACATTCCCACC ACGTTGGATGTAAAGTCTCTGCGTTCTGGC
rs2071351 33045675 ACGTTGGATGTTACTGATGGTGCTGCTCAC
ACGTTGGATGAATTGTTCCCTGAGCCAGAC rs3117227 33058913
ACGTTGGATGCACAGTTCCCTAACGAGAAG ACGTTGGATGGGTACCCCTTGATAACCATC
rs2144014 33067793 ACGTTGGATGTGTGTAGATCTCTAGCGAGG
ACGTTGGATGAAGCCTCCAAGAAATTTGGG rs3130216 33079418
ACGTTGGATGAGAGACTGAGTTCAGTGTGG ACGTTGGATGACTGAGACCACCCATCATAC
rs1883414 33089789 ACGTTGGATGCAATCCATTGGTGTAACAGG
ACGTTGGATGAGATTACCACCTATAGACTG rs3129272 33099767
ACGTTGGATGTCCACTCCACAGATGATGAG ACGTTGGATGTGTTCTTCCTAGAGGCACAG
rs2294479 33101481 ACGTTGGATGACCTCAGTTTTGCATCCTGC
ACGTTGGATGTCCATTTTTGTCCCCTGGAC rs2294478 33102058
ACGTTGGATGTTTTGTCCCCCATCCCTTTC ACGTTGGATGACAAGAAGGAGATGGTCTGG
rs2015610 33110987 ACGTTGGATGCTCAGTGATTGGCACAAGTG
ACGTTGGATGGCCTAAAGGTTTCTCTGTAC rs3130153 33119103
ACGTTGGATGGGTACCATCAGAATACTGTC ACGTTGGATGTTCACGGCTTGACTCAATGG
rs3129206 33128803 ACGTTGGATGCTAAGGGAAGGAGAACTCTC
ACGTTGGATGAAGGTGGCACTGATTCTAGC rs734181 33133246
ACGTTGGATGTAGACTGGGCTATGTAGCAC ACGTTGGATGATGGCTCCAGTTTCTGACAC
rs2076311 33148710 ACGTTGGATGATCCCACCCCCATTCTTATC
ACGTTGGATGAAGAAGGCAAGAGCAGGAAG rs2855457 33158566
ACGTTGGATGCCAAGCCAGTCAACATTTTC ACGTTGGATGTTGTCTCATTTCCAGAGCCC
rs2855433 33161160 ACGTTGGATGGGTTTAGGAGATGAGTTGGG
ACGTTGGATGATCCACAGATGTGTGCTCAG rs2982275 33168406
ACGTTGGATGCCTTCTTCTGTGTCTCCATC ACGTTGGATGAAGTGGGTGTTTTGACCAAG
rs421446 33178124 ACGTTGGATGACTGTGTATGCGTGACACTC
ACGTTGGATGTGCAGAACCAGTGGAAAGGG rs1704996 33185888 rs213213 33186822
rs213194 33198941 ACGTTGGATGGGTGGAGAGATGTGATTTCC
ACGTTGGATGTATACCGTCCAATAGGAGGC rs213224 33209549
ACGTTGGATGATCCAAGCACTTTGGGAAGC ACGTTGGATGTGTTTTGCCATGTTGGCCAG
rs213225 33211713 rs213226 33212398 ACGTTGGATGCCTGGCTTGCTTTCTTCTTG
ACGTTGGATGGCAGCATGGTTTTGTACAAG rs105445 33220331
ACGTTGGATGTGAGGGAACGCATAGCGCAG ACGTTGGATGTTAACTGACCTCGCCCTTGC
rs1269806 33229599 ACGTTGGATGAACACAGAAAGACCCTCATC
ACGTTGGATGGAGTTCTTTGCATCATCTAC rs213202 33235198
ACGTTGGATGTCACCATGAGTTTCACCACC ACGTTGGATGGCTCTAAGCATCATTGTGGG
rs2231260 33249259 ACGTTGGATGAGGCCACTGCTCCTCTGATAC
ACGTTGGATGTGCCTCTTCTGTACTTGGGC rs464865 33259080
ACGTTGGATGGCAGCTTATGCAAGAGTGAC ACGTTGGATGAAAAGAAACCGCACCGCTAC
rs1014779 33278611 ACGTTGGATGGCCAAGGACGGCTTGGAATA
ACGTTGGATGACGAACACTAACGATGGCTG rs1061783 33284571
ACGTTGGATGCCTTTATCTCTGTGGACTTG ACGTTGGATGAGTTCTGGAAATACCTTGGG
rs3130016 33308361 ACGTTGGATGAGTGGCTCATGCCTGTAATC
ACGTTGGATGCTCCTGACTTTAGGTGATCC rs3130267 33318929
ACGTTGGATGACCATGTGGAGCAAAAAGGC ACGTTGGATGAACACGTTGGATGTTGTGCG
rs1265492 33322652 ACGTTGGATGAGGCCCTCAAAATCACAAAC
ACGTTGGATGACGTGTTAGATGTGAGGAAG rs3117323 33327502
ACGTTGGATGCACGTGTTTATCTGCTGACC ACGTTGGATGTTGGGTGTTTCTCAGAGAGG
rs211450 33335443 ACGTTGGATGATGTCTGGAACTGGACCCTG
ACGTTGGATGATGGTGCCCGTACGGTTTGG rs211447 33346185
ACGTTGGATGTTTTGTCGACCCTGCACTTG ACGTTGGATGGCCATAAGATCAGATGAGCC
rs465877 33358320 ACGTTGGATGAGGAGAATGGCATGAACCCC
ACGTTGGATGTTTTTGAGACAGAGTCTGGC rs456993 33360326
ACGTTGGATGCCAACCTTTGATATCCTGGG ACGTTGGATGTCCCCCTTTACCTTCCATTG
rs211457 33367887 ACGTTGGATGCCAGACAGCATGAACAGAAG
ACGTTGGATGAAGTCCAGCTTTTCCCCTAG rs3106193 33377160
ACGTTGGATGCCGAAGTGTTCGGATTATAG ACGTTGGATGGAAAAGGTGACTAAAAGGTC
rs1705003 33388001 ACGTTGGATGAGTAGAAGAAGGACCACCTG
ACGTTGGATGATTGGTCAAGTCTCCCATGG rs2076775 33396500
ACGTTGGATGTCACCCTTTCTCTTTCCTCC ACGTTGGATGTGGGAGTCTGATGGACTTTC
rs453590 33405670 ACGTTGGATGAACTCACTTTCCTCCCTAGG
ACGTTGGATGAAGCCCAACAAGGTATTGGG rs3119027 33416775
ACGTTGGATGAGCATCATTGGCAGGTGAGG ACGTTGGATGAGTTTGGGGATGGGAGTCAG
rs3119025 33425122 ACGTTGGATGAGAACTTTCCCTCTAGCCTG
ACGTTGGATGATCGAGTTCCCACAGCATAG rs1755047 33433266
ACGTTGGATGTCTCTCTTTCCCTGTAGCTC ACGTTGGATGATGCCCAAGCCAAGAAAGAG
rs1755049 33440490 ACGTTGGATGTTCAGCCTCTGGTGTAGCTG
ACGTTGGATGTAACACGGTGAAACCCCGTC rs2772381 33456461 rs210190 33466198
ACGTTGGATGATAGTGCTCACTGGCTGAAG ACGTTGGATGACTATCACAGGAAGCAGTCG
rs1755038 33467280 ACGTTGGATGATGATGGCAGCAGCCACTGC
ACGTTGGATGCCTCCAGTAGCATGTAAGGC rs769051 33476004
ACGTTGGATGACCTCTGCAGACTTAGACTG ACGTTGGATGCGCATAAAGTAGAGGGACTC
rs210180 33487120 ACGTTGGATGAAACCCACACCTGCAGTGAG
ACGTTGGATGAGGCTTTCCACACACTCCTG rs210184 33488865
ACGTTGGATGTCCTTTCCTCTGGGTTAGCC
ACGTTGGATGTGTCTTCACCACAGGCAGTG rs429789 33497846
ACGTTGGATGTTTGAGATGGAGTCTCGCTC ACGTTGGATGAGGAGAATTGCTTGAACCCG
rs210196 33509584 ACGTTGGATGAAGCAGCTGGGAAAGAAACG
ACGTTGGATGATGACAGTGCTTCCAGAGAG rs210203 33513090
ACGTTGGATGAGGGCAAATGAAATCTGTCC ACGTTGGATGTCTCTGTGCCTATGCATACC
rs210158 33521419 ACGTTGGATGCCCTTGCCTTATCTTTCTTG
ACGTTGGATGCACTAGGGAAATGGTTGTGC rs210169 33526576 rs210131 33537576
ACGTTGGATGAGAGGAGGAAGAGCTGAAAG ACGTTGGATGATTCATGTAAGGCACGGACC
rs210133 33538658 ACGTTGGATGGGCTCACCAACCTTCTCATG
ACGTTGGATGTAACTGGATAAGCTGCCCTC rs210132 33538781
ACGTTGGATGTTTCAGAGACACCAGACATG ACGTTGGATGTTGCTAAAGTCTCAGGTGGG
rs210135 33542803 ACGTTGGATGAGAACCCTCCAGATGAACTC
ACGTTGGATGACTACAGGGCTTAGGACTTG rs513349 33543830
ACGTTGGATGCTTCCTCGGGTTCCTATATC ACGTTGGATGGAGAAACAAGGTGGTCACAG
rs210139 33545520 ACGTTGGATGTCTAAGACATGAGTGCTGGG
ACGTTGGATGTTTTATGTTGGGCTCCCACC rs210141 33548935
ACGTTGGATGCCAAGAGCTCTCAAGAAGGG ACGTTGGATGTAACAAGGCCTTGCCCCTAG
rs210145 33549551 ACGTTGGATGTGGCCTAAATTCCCGGTGAG
ACGTTGGATGAACATCCCTAGACTGGGTCC rs2894350 33556912
ACGTTGGATGTCTAAATTCAGGACCCTGGC ACGTTGGATGCAGAGAGACTGATGGAGAAG
rs396746 33558906 ACGTTGGATGTTTGGCCTCATTGTTGGCTG
ACGTTGGATGAAGACCTCAGATAGACTGGG rs210162 33561565
ACGTTGGATGTTCGACTCTTCCCGGACTCC ACGTTGGATGTTCCCCCTTGCCCCATATGAG
rs210120 33576523 ACGTTGGATGGCTCAGCTTAAATGTCTCCC
ACGTTGGATGGAGAGCAGAAAAGAGAGAGG rs407415 33581077
ACGTTGGATGAACCCACCTTTCTTGTTGGC ACGTTGGATGTCTTCCTTTCTCCAGACTCC
rs2395451 33589837 ACGTTGGATGGGACTGGATGAGGTTTTTTA
ACGTTGGATGGCTAAGTACCGTGTTTTACAG rs1536043 33594867
ACGTTGGATGTCCTCCACTTTTTGTTGGCC ACGTTGGATGTTATCCCTTACCCTAGGTGG
rs1536042 33620471 ACGTTGGATGAGTCACTTGAATGGGCTCAG
ACGTTGGATGTAACAGCCCTTATAGGGTGG rs999943 33626118
ACGTTGGATGTATAGCTGTGGACTGGGCTG ACGTTGGATGAGGAAGGAAGCCTGTTGCAG
rs2229634 33640290 ACGTTGGATGGCCATGCACAGGAAAATCAG
ACGTTGGATGTACCAGCTGAAGCTCTTTGC rs753890 33654495
ACGTTGGATGTCACGTGGTCCTTTCATCAC ACGTTGGATGTGAGAAGAGTGGGCATGATG
rs658087 33667130 ACGTTGGATGAAGCAGGCTTTTTGCCTCTC
ACGTTGGATGGGGAGGAAGCCAAAAATAGC rs2281829 33677752
ACGTTGGATGGCTGTAGGAAACACGTGTTC ACGTTGGATGTCCTCTTACACCCCATATCC
rs1555965 33679261 ACGTTGGATGCTTTACACTTTGGGCCAGTG
ACGTTGGATGTGGCCCAGGTTATACGATAC rs549652 33688213
ACGTTGGATGAATGTCATCAGGAAGCCCTG ACGTTGGATGCCTGCGTGGTTTAAAAGCTC
rs630792 33692421 ACGTTGGATGTCTCCGATGAGCAGCATTAG
ACGTTGGATGGCTGAACAAATCAGCTCTGG rs597723 33696190
ACGTTGGATGCCTTCCATCCCTCCAAATTC ACGTTGGATGCACACTTCCCTCTCACTGTG
rs608971 33703990 ACGTTGGATGTAGAATCCGGCGTGTATGTG
ACGTTGGATGTTCTGATTCACAGGTCTGGC rs568901 33712149
ACGTTGGATGAAGTCGACGCCCATTCTGAC ACGTTGGATGAGACAGCCAGAGCAACTCAG
rs570749 33712340 ACGTTGGATGAGCACTTCGTTAGACACTGC
ACGTTGGATGTTTCAGAAGCTGCACCTGAC rs530614 33716891
ACGTTGGATGTCTCTCCTCCTCTTTGTCCC ACGTTGGATGGACTGGCATGTCCAGCTGTC
rs2395449 33730616 ACGTTGGATGAGACTACGCATCCTCTTCTC
ACGTTGGATGTGCAAACCTCTCAGAGTCTC rs755496 33736487
ACGTTGGATGCCTGTGCCCTTATGATTCTG ACGTTGGATGAACCAATCCCTGGGATGGAG
rs755497 33736530 ACGTTGGATGAAGCATGGTTCTGTGCCCTG
ACGTTGGATGAACCAATCCCTGGGATGGAG rs943473 33745761
ACGTTGGATGATGCTGAGAGCTCACCCTTG ACGTTGGATGTTTGCATCTGTGGAGAGCTC
rs943474 33751945 ACGTTGGATGACTTTGAAACTGAACTGAAG
ACGTTGGATGCTTTGAAGTAGGAGGATCTG rs943475 33752089
ACGTTGGATGAACTGGATTGGGAAAGGGAG ACGTTGGATGTTTCCCACTAGGACTCTTCC
rs943479 33753874 ACGTTGGATGTTAGTTACACTGCTGCTGGC
ACGTTGGATGGACTCGGCTTCTGAATATGC rs2395402 33755534
ACGTTGGATGCCCTGCATTGACTGTCTTAC ACGTTGGATGAACTATGACACACCCGAAGC
rs2013365 33765798 rs2894342 33776504
ACGTTGGATGAATAGCTGTGTGACCTTGGG ACGTTGGATGTTACAACAGCCCTGAGGTTC
rs1547668 33777490 ACGTTGGATGTAGTGGCTGTTTCTCTCCTG
ACGTTGGATGATATCCGTGGCAATTCCCAC
[0027] Genotyping HLA Loci
[0028] The invention features a novel method of genotyping Human
Leukocyte Antigen (HLA) genes using patterns of neighboring single
nucleotide polymorphisms (SNPs). The SNP-based method is an
improvement over existing hybridization-based techniques, as it
allows quick and inexpensive genotyping of the HLA loci. This
method does not directly assess the intra-gene variation, as is
done by all other current methods for HLA genotyping, but rather
defines HLA genotypes by studying the neighboring extra-genic
variation(s) which, due to LD patterns, is conveniently linked to
the HLA loci. By "extra-genic" herein is meant outside or in the
neighboring region(s) of the HLA allele to be genotyped.
Identification of the correlation of this extra-genic variation to
the HLA gene alleles allows for the discovery and utilization of
surrogate markers for HLA genotypes.
[0029] One aspect of the invention provides a method of genotyping
an HLA gene, such as for example an HLA-A or an HLA-DRB1 gene. The
method comprises determining the nucleotide present at one or more
extra-genic SNP sites, wherein the SNP is associated with an HLA
genotype. For example, to genotype the HLA-A allele, an extra-genic
SNP to be assessed can be rs2517862, rs1655930, rs1616549,
rs376253, rs1961135, rs2517706, rs2517701, rs2517699, rs435766,
rs410909, rs2394255, rs1264807, rs2530388, rs356963, rs2286405,
rs2240619, rs3129012, rs259938, or any combination thereof. Another
example involves genotyping the HLA-DRB allele, wherein an
extra-genic SNP to be assessed can be rs742697, rs523627,
rs3129960, rs2395163, rs2395165, rs983561, rs2239804, rs2213584,
rs2395182, rs2858860, rs3129907, rs1059544, rs1987529, or any
combination thereof.
[0030] Nomenclature and designations of the HLA alleles have been
described by Marsh et al., Tissue Antigens (2002) 60:407-464. A
summary of HLA-A, -B, -C, -DRB1/3/4/5, -DQB1 alleles and their
association with serologically defined HLA-A, -B, -C, -DR and -DQ
antigens is provided by Schreuder et al., Tissue Antigens (2001)
58:109-140.
[0031] Methods of determining or analyzing SNPs are known in the
art. For example, to detect any particular SNP in target DNA
sample, e.g., a DNA sample from a subject to be tested, preferable
a human subject, one can employ any of the known procedures in the
art. For example, two distinct types of analysis and seven
procedures are described in U.S. patent application Ser. No.
10/213,272, Publication No. 20030170665, incorporated herein by
reference in its entirety. The first type of analysis is sometimes
referred to as de novo characterization. This analysis compares
target sequences in different individuals to identify points of
variation, i.e., polymorphic sites. By analyzing a group of
individuals representing the greatest variety patterns
characteristic of the most common alleles/haplotypes of the locus
can be identified, and the frequencies of such populations in the
population determined. Additional allelic frequencies can be
determined for subpopulations characterized by criteria such as
geography, race, or gender. The second type of analysis determines
which form(s) of a characterized polymorphism are present in
individuals under assessment. There are a variety of suitable
procedures:
[0032] 1). Allele-Specific Probes
[0033] The design and use of allele-specific probes for analyzing
SNPs is described by e.g., Saiki et al., Nature 324:163-166 (1986);
Dattagupta, EP 235,726, Saiki, WO 89/11548. Allele-specific probes
can be designed that hybridize to a segment of target DNA from one
individual but do not hybridize to the corresponding segment from
another individual due to the presence of different polymorphic
forms in the respective segments from the two individuals.
Hybridization conditions should be sufficiently stringent that
there is a significant difference in hybridization intensity
between alleles, and preferably an essentially binary response,
whereby a probe hybridizes to only one of the alleles. Some probes
are designed to hybridize to a segment of target DNA such that the
polymorphic site aligns with a central position (e.g., in a 15 mer
at the 7 position; in a 16 mer, at either the 8 or 9 position) of
the probe. This design of probe achieves good discrimination in
hybridization between different allelic forms.
[0034] Allele-specific probes are often used in pairs, one member
of a pair showing a perfect match to a reference form of a target
sequence and the other member showing a perfect match to a variant
form. Several pairs of probes can then be immobilized on the same
support for simultaneous analysis of multiple polymorphisms within
the same target sequence.
[0035] 2). Tiling Arrays
[0036] The SNPs can also be identified by hybridization to nucleic
acid arrays. Subarrays that are optimized for detection of a
variant forms of a precharacterized polymorphism can also be
utilized. Such a subarray contains probes designed to be
complementary to a second reference sequence, which is an allelic
variant of the first reference sequence. The inclusion of a second
group (or further groups) can be particular useful for analyzing
short subsequences of the primary reference sequence in which
multiple mutations are expected to occur within a short distance
commensurate with the length of the probes (i.e., two or more
mutations within 9 to 21 bases).
[0037] 3). Allele-Specific Primers
[0038] An allele-specific primer hybridizes to a site on target DNA
overlapping an SNP and only primes amplification of an allelic form
to which the primer exhibits perfect complementarily. See Gibbs,
Nucleic Acid Res. 17, 2427-2448 (1989). This primer is used in
conjunction with a second primer which hybridizes at a distal site.
Amplification proceeds from the two primers leading to a detectable
product signifying the particular allelic form is present. A
control is usually performed with a second pair of primers, one of
which shows a single base mismatch at the polymorphic site and the
other of which exhibits perfect complementarily to a distal site.
The single-base mismatch prevents amplification and no detectable
product is formed. The method works best when the mismatch is
included in the 3'-most position of the oligonucleotide aligned
with the polymorphism because this position is most destabilizing
to elongation from the primer.
[0039] 4). Direct-Sequencing
[0040] The direct analysis of the sequence of any samples for use
with the present invention can be accomplished using either the
dideoxy-chain termination method or the Maxam-Gilbert method (see
Sambrook et al., Molecular Cloning, A Laboratory Manual (2nd Ed.,
CSHP, New York 1989); Zyskind et al., Recombinant DNA Laboratory
Manual, (Acad. Press, 1988)).
[0041] 5). Denaturing Gradient Gel Electrophoresis
[0042] Amplification products generated using the polymerase chain
reaction can be analyzed by the use of denaturing gradient gel
electrophoresis. Different alleles can be identified based on the
different sequence-dependent melting properties and electrophoretic
migration of DNA in solution. Erlich, ed., PCR Technology,
Principles and Applications for DNA Amplification, (W. H. Freeman
and Co, New York, 1992), Chapter 7.
[0043] 6). Single-Strand Conformation Polymorphism Analysis
[0044] Alleles of target sequences can be differentiated using
single-strand conformation polymorphism analysis, which identifies
base differences by alteration in electrophoretic migration of
single stranded PCR products, as described in Orita et al., Proc.
Nat. Acad. Sci. 86, 2766-2770 (1989). Amplified PCR products can be
generated as described above, and heated or otherwise denatured, to
form single stranded amplification products. Single-stranded
nucleic acids may refold or form secondary structures which are
partially dependent on the base sequence. The different
electrophoretic mobilities of single-stranded amplification
products can be related to base-sequence difference between alleles
of target sequences.
[0045] 7). Single Base Extension
[0046] An alternative method for identifying and analyzing SNPs is
based on single-base extension (SBE) of a fluorescently-labeled
primer coupled with fluorescence resonance energy transfer (FRET)
between the label of the added base and the label of the primer.
Typically, the method, such as that described by Chen et al., (PNAS
94:10756-61 (1997)), uses a locus-specific oligonucleotide primer
labeled on the 5' terminus with 5-carboxyfluorescein (FAM). This
labeled primer is designed so that the 3' end is immediately
adjacent to the polymorphic site of interest. The labeled primer is
hybridized to the locus, and single base extension of the labeled
primer is performed with fluorescently-labeled
dideoxyribonucleotides (ddNTPs) in dye-terminator sequencing the
effect of mtDNA D-loop sequence polymorphism on milk production,
each cow was the next generation of the herd.
[0047] TABLE 3 shows exemplary extra-genic SNPs that correspond to
HLA-A alleles and can be used in genotyping HLA-A alleles. The SNPs
and HLA-A allele are lined up in each row of the table from the
left to the right according to their respective positions on
chromosome 6. The percentage numbers on the right column represent
the likelihood of the identity of a particular HLA-A allele when
the exemplary SNPs are determined to be as shown in the respective
rows. For example, in row 1, the HLA-A allele has a 100% likelihood
to be HLA-A*2402, when the 18 SNPs listed are determined to be the
respective nucleotides as shown in row 1. Take row 5 as another
example, the HLA-A allele has a 92% likelihood to be HLA-A*101,
when the 18 SNPs listed are determined to be the respective
nucleotides as shown in row 5. The allele-type determinative SNPs
between HLA-A*2402 and HLA-A*101 include: rs2517862, rs1655930,
rs376253, rs1961135, rs2517706, rs1264807, and rs3129012.
3TABLE 3 1 2 Legend: 1 = A; 2 = C; 3 = G; 4 = T
[0048]
4TABLE 4 3 Legend: 1 = A; 2 = C; 3 = G; 4 = T
[0049] The above table shows exemplary extra-genic SNPs that
correspond to HLA-DRB1 alleles and can be employed to genotype
HLA-DRB1 alleles. Again the relative positions of the SNPs and the
HLA-DRB1 on human chromosome 6 are shown (from left to right in
each row). The letters on the right-most column are arbitrarily
assigned to the SNP-haplotype alleles as shown on each row. For
example, row 1 corresponds to SNP-haplotype allele J, with the
extra-genic SNPs determined to be the nucleotides as shown in this
row. Where ambiguity exists, e.g., row 4, where the SNP-haplotype
could be B or W, and this ambiguity may be resolved by determining
an additional SNP: rs3129907. And if rs3129907 is 1 or A, the
SNP-haplotype allele will be B, and if rs3129907 is 3 or 6, the
SNP-haplotype allele will be W. Similarly, row 6, the SNP-haplotype
allele can be ascertained by determining the SN-P rs1059544 (2 or C
will correspond to SNP-haplotype allele U, and 4 or T will
correspond to SNP-haplotype allele V). Also, row 11, the
SNP-haplotype allele can be ascertained by determining the SNP
rs1987529 (3 or 6 will correspond to SNP-haplotype allele K, and 1
or A will correspond to SNP-haplotype allele T). Also, Similarly,
row 14, the SNP-haplotype allele can be ascertained by determining
the SNP rs1987529 (1 or A will correspond to SNP-haplotype allele
G, and 3 or G will correspond to SNP-haplotype allele H).
Similarly, row 16, the SNP-haplotype allele can be ascertained by
determining the SNP rs2395165 (4 or T will correspond to
SNP-haplotype allele A, and 2 or C will correspond to SNP-haplotype
allele R).
[0050] Next, FIG. 4B shows the percentage of a particular SNP
haplotype allele that bears the indicated HLA allele. For example,
SNP-haplotype allele J, having the SNPs as shown in row 1 above,
corresponds to an HLA-DRB1 allele that has a 100% likelihood to be
HLA-DRB1*1302. Take row 2 as another example, SNP-haplotype allele
N, having the SNPs as shown in this row, corresponds to an HLA-DRB1
allele that has a 92.6% likelihood to be HLA-DRB1*1501.
[0051] The invention further features a method of predicting or
assisting in the prediction of the likelihood or probability of
development of a disease, particularly an MHC-linked disease, in a
subject, preferably a human subject. The method comprises
genotyping an HLA gene in the subject to be tested by determining
the nucleotide present at one or more extra-genic SNP sites,
wherein the SNP is associated with an HLA genotype. MHC-linked
diseases include, but are not limited to, ankylosing spondylitis,
Behcet Syndrome, common variable immunodeficiency, Goodpasture
Syndrome, psoriasis, inflammatory bowel disease, insulin-dependent
diabetes mellitus (type 1), multiple sclerosis, myasthenia gravis,
pemphigus vulgaris, rheumatoid arthritis, systemic lupus
erythematosus. Identification of an HLA genotype in the subject
which is associated with a disease is indicative that the subject
has a greater likelihood of developing the disease. For example,
HLA-DRB1*1101 genotype is associated with pemphigoid diseases, as
discussed above.
[0052] The invention further features a method of predicting or
assisting in the prediction of the likelihood or probability of
development of a disease, particularly an autoimmune disease, in a
subject, preferably a human subject. The method comprises
genotyping an HLA gene in the subject to be tested by determining
the nucleotide present at one or more extra-genic SNP sites,
wherein the SNP is associated with an HLA genotype. Identification
of an HLA genotype in the subject which is associated with a
disease is indicative that the subject has a greater likelihood of
developing the disease. For example, HLA-DR2 haplotype is linked or
associated with multiple sclerosis and lupus. Examples of
autoimmune diseases grouped based on main target organs include,
but are not limited to:
[0053] 1) Nervous System: multiple sclerosis, myasthenia gravis,
autoimmune neuropathies such as Guillain-Barr, autoimmune
uveitis;
[0054] 2) Gastrointestinal System: Crohn's Disease, ulcerative
colitis, primary biliary cirrhosis, autoimmune hepatitis;
[0055] 3) Blood: autoimmune hemolytic anemia, pernicious anemia,
autoimmune thrombocytopenia;
[0056] 4) Endocrine Glands: Type 1 or immune-mediated diabetes
mellitus, Grave's Disease, Hashimoto's thyroiditis, autoimmune
oophoritis and orchitis, autoimmune disease of the adrenal
gland;
[0057] 5) Blood Vessels: temporal arteritis, anti-phospholipid
syndrome, vasculitides such as Wegener's granulomatosis, Behcet's
disease;
[0058] 6) Multiple Organs Including the Musculoskeletal System
(These diseases are also called connective tissue (muscle,
skeleton, tendons, fascia, etc.) diseases.): rheumatoid arthritis,
systemic lupus erythematosus, scleroderma, polymyositis,
dermatomyositis, spondyloarthropathies such as ankylosing
spondylitis, Sjogren's syndrome;
[0059] 7) Skin: psoriasis, dermatitis herpetiformis, pemphigus
vulgaris, vitiligo.
[0060] A further aspect of the invention provides a method of
predicting or assisting in the prediction of the likelihood of
developing an immune response in a subject, preferably a human
subject. An immune response may be developed against an infecting
organism or agent. Alternatively, an immune response may comprise a
host-graft response, e.g., rejection of organ transplants. The
method comprises genotyping an HLA gene in the subject to be tested
by determining the nucleotide present at one or more extra-genic
SNP sites, wherein the SNP is associated with an HLA genotype. The
method may also comprise separately genotyping an HLA gene in a
host (e.g., a blood or organ recipient or donee) and the same HLA
gene (or the corresponding HLA gene) in a graft (e.g., a blood or
organ donor) by determining the nucleotide present at one or more
extra-genic SNP sites in the host and the graft, wherein the SNP is
associated with an HLA genotype. Genotyping an HLA gene in a host
may involve assessing more, fewer, or the same extra-genic SNPs as
compared the extra-genic SNP(s) to be assessed in a graft.
[0061] In preferred embodiments of the invention, more than one
extra-genic SNP, more preferably more than three extra-genic SNPs,
more preferably more than five extra-genic SNPs, and more
preferably more than seven extra-genic SNPs are determined in order
to determine the genotype of an HLA allele.
[0062] An exemplary method of determining whether or not a host and
a graft have the same HLA alleles or immune-compatible HLA alleles
may include:
[0063] a) determining the HLA allele in the host (or graft) by
ascertaining the nucleotide present at one or more extra-genic SNP
sites or any other method;
[0064] b) selecting extra-genic SNPs to be assessed in the graft
(or host) based on the HLA allele identity as determined in a);
[0065] c) assessing the selected extra-genic SNPs to identify the
HLA allele genotype.
[0066] For example, if a host is determined by a method of the
invention or any other method to have an HLA-A*101 allele (e.g.,
having SNP-haplotype as shown in row 5 of TABLE 3 above), only
rs2517862 and/or rs1655930 need to be assessed to ascertain that a
graft does not have HLA-A*101. Based on the information in TABLE 3,
one can optimize the selection of SNPs to be assessed.
[0067] All publications, patents, patent applications and
information from databases cited above are hereby incorporated by
reference in their entirety for all purposes to the same extent as
if each individual publication or patent application were
specifically and individually indicated to be so incorporated by
reference.
[0068] The invention is now further described in the following
non-limiting examples.
EXEMPLIFICATION
Example 1
Materials and Methods
[0069] DNA Samples
[0070] Samples were obtained from the Coriell Cell Repository and
drawn from the collection of Utah CEPH pedigrees of European
descent. One hundred thirty-six independent, grandparental
chromosomes were used for haplotype construction. Of these
chromosomes, 96 were in common with Gabriel et al. (2002) and,
therefore, were used for comparison with the genome-wide LD
structure. Identifiers for all individuals can be found at the
Inflammatory Disease Research Group (IDRG) Website.
[0071] Genotyping and Data Checking
[0072] All SNPs for which genotyping was attempted were publicly
available at the dbSNP Web site. SNPs were selected mainly to
achieve a desired spacing (1/20 kb); however, SNPs with more than
one submitter were preferentially chosen. SNP primers and probes
were designed in multiplex format (average fivefold multiplexing)
with SpectroDESIGNER software (Sequenom). A total of 435 assays
were designed. Assays were considered successful and genotype data
were included in the analyses described herein if they passed all
of the following criteria: (1) a minimum of 75% of all genotyping
calls were obtained, (2) markers did not deviate from
Hardy-Weinberg equilibrium, and (3) markers had no more than one
Mendelian error. These criteria defined 201 successful assays.
Genotype calls for successful markers were then set to zero for any
single Mendelian error. All of these working assays had minor
allele frequencies 15%, and 89% of these assays had minor allele
frequencies 110%. Overall, for successful markers, 97.6% of all
attempted genotypes were obtained. The entire list of SNP assays,
as well as detailed genotyping information, can be found at the
IDRGWeb site. Four-digit HLA types were determined for HLA-A,
HLA-B, HLA-C, HLA-DRB1, HLA-DMB1, HLADQA1, HLA-DQB1, HLA-DPA1, and
HLA-DPB1, as described elsewhere (Begovich et al. 1992; Carrington
et al. 1994, 1999; Moonsamy et al. 1997; Bugawan et al. 2000).
Typing was performed twice independently, and conflicting types
were resolved, in most cases, by two independent retyping
experiments. TAP1 and TAP2 were genotyped as described elsewhere
(Carrington et al. 1993). D6S2971, D6S2749, D6S2874, D6S273,
D6S2876, D6S2751, D6S2741, and D6S2739 were typed as described
elsewhere (Martin et al. 1998). Genotyping details for the 11
remaining microsatellites can be found as supplemental information
on the IDRGWeb site. D6S2972 and D6S265 genotypes were typed twice
(IDRG; Martin et al. 1998), and conflicts were resolved by
retyping. Alias details for all microsatellites are provided
elsewhere (Cullen et al. 2003).
[0073] Genotyping Details
[0074] Multiplex PCR was performed in six microliter volumes
containing 0.1 units of Taq polymerase (Amplitaq Gold, Applied
Biosystems), 5 ng genomic DNA, 2.5 pmol of each PCR primer, and 2.5
lmol of dNTP. Thermocycling was at 95.degree. C. for 15 minutes
followed by 45 cycles of 95.degree. C. for 20s, 56.degree. C. for
30s, 72.degree. C. for 30s. Unincorporated dNTPs were deactivated
using 0.3U of Shrimp Alkaline Phosphatase (Roche) followed by
primer extension using 5.4 pmol of each primer extension probe, 50
.mu.mole of the appropriate dNTP/ddNTP combination, and 0.5 units
of Thermosequenase (Amersham Pharmacia). Reactions were heated to
94.degree. C. for 2 minutes, followed by 40 cycles of 94.degree. C.
for 5 s, 50.degree. C. for 5 s, 72.degree. C. for 5 s. Following
addition of a cation exchange resin to remove residual salt from
the reactions, seven nanoliters of the purified primer extension
reaction was loaded onto a matrix pad (3-hydroxypicoloinic acid) of
a SpectroCHIP (Sequenom, San Diego, Calif.). SpectroCHIPs were
analyzed using a Bruker Biflex III MALDI-TOF mass spectrometer
(SpectroREADER, Sequenom, San Diego, Calif.) and spectra processed
using SpectroTYPER (Sequenom).
[0075] Eleven of the microsatellites were amplified using the
following primers/amplification programs:
5 Forward Primer (5' to 3') Reverse Primer (5' to 3') PCR
Microsatellite (SEQ ID NOS: 1279-1289) (SEQ ID NOS: 1290-1300)
program D6S1542 ACTGGGTGCATCAGGGAG CTTTACAACCCTTGGCAGC EPA D6S1560
CTCCAGTCCCCACTGC CCCAAGGCCACATAGC 64ANN D6S1701 GGTGTCAGAGCAANATTCC
AACAAAGTATCACAAACTGGGAG RG-MSATS D6S2747 GGAGACACATTCAAACCATAGG
CAATTGGTGACATACATCAACTTG MSATTD D6S2896 AATGGCTGTTAGGAAGAAGC
TCTTCCTTAGCTGCTGCTG MSATTD D6S2793 AATAGCCATGAGAAGCTATGTGGGGGAG
CTACCTCCTTGCCAAAGTTGCTGTTTGTG RG-MSATS D6S2814 GTGAAATCAGCCTGCTTCTG
GAACACAACCATCTCTGCTC RG-MSATS D6S2840 AGATGGCATTTGGAGAGTGCAG
TCCTTACAGCAGAGATATGTGG RG-MSATS D6S265 ACGTTCGTACCCATTAACCT
ATCGAGGTAAACAGCAGAAA RG-MSATS D6S2972 GAAATGTGAGAATAAAGGAGA
GATAAAGGGGAACTACTACA EPA D6S258 GCAAATCAAGAATGTAATTCCC
CTTCCAATCCATAAGCATGG MSATJH
[0076] The forward primers were fluorescently tagged with 6-FAM,
TET or HEX. Amplification was performed in 15 microliter volumes
containing 0.8 units Taq polymerase (Roche Applied Science), 25 ng
DNA, 200 .mu.M dNTPs, 2.4 pmol each primer, 3.0 mmol dNTPs, and
1.times.PCR buffer (1.5 mM MgCl2, 10 mM Tris-HCl, 50 mM KCl, pH
8.3, Roche Applied Science). Reactions were run in one of the
following MJ Research thermocyclers (PTC-100, PTC-200 or Genomyx
CycLR). Samples were then multiplexed and 2-3 .mu.l of multiplex
was combined with an equal amount of size standard loading buffer
mix (containing formamide, blue dextran and fluorescently labeled
size standard Genescan-350 or -500 Tamara), denatured for 3 minutes
at 95.degree. C., and electrophoresed on a 5% gel (National
Diagnostics), using an ABI model 377 DNA sequencer (Applied
Biosystems).
[0077] Genotypes for individuals from families 1331, 1332, 1347,
1362, 1413, 1416 and 884 for D6S1542, D6S1560, D6S1701, D6S1666,
D6S265, D6S258 were obtained from the CEPH website
(http://www.cephb.fr/test/cephdb/). To ensure correspondence in
allele sizes with those genotyped for this study, individual 1347-2
was genotyped for these loci.
[0078] For RG-MSATS amplification, reactions were heated to
95.degree. C. for 2 minutes followed by 29 cycles of 94.degree. C.
for 45 s, 57.degree. C. for 45 s, 72.degree. C. for 1 minute. The
final extension was at 72.degree. C. for 7 minutes. MSATJH and
64ANN were the same as RG-MSATS, except annealing was carried out
at 55.degree. C. and 64.degree. C. respectively. MSATTD was a
touchdown annealing starting at 60.degree. C. and decreasing in
each subsequent cycle by 0.3.degree. C. until arriving at
55.degree. C. were annealing was held constant for the remaining 15
cycles. EPA reactions were heated to 95.degree. C. for 2 minutes
followed by 4 cycles at 96.degree. C. for 30 s, 57.degree. C. for
90 s and 72.degree. C. for 90 s; followed by 28 cycles at
95.degree. C. for 30 s, 55.degree. C. for 45 s, 72.degree. C. for 1
minute. The final extension was at 72.degree. C. for 30
minutes.
[0079] D'Confidence Limits, Definition of Haplotype Blocks, and
Structure Comparison
[0080] Pairwise D' values--estimates of the strength of LD
(Lewontin 1964)--for SNP markers were assessed and haplotype blocks
were defined as per Gabriel et al. (2002). In brief, D' confidence
limits were determined by calculating the probability of the
observed data for all possible values of D', from which an overall
probability distribution was determined. For all blocks identified,
the outermost marker pair was required to be in strong LD, with an
upper confidence limit (CU)>0.98 and a lower confidence limit
(CL)>0.7. Blocks defined by only two markers required confidence
bounds of (CL)>0.8 and (CU)>0.98 and an intervening distance
of =<20 kb; for three consecutive markers, all pairs had to have
confidence bounds of CL>0.5 and CU>0.98 and an intervening
distance of <30 kb; and for four markers, the fraction of
informative pairs in strong LD (CL>0.7 and CU>0.98) was
required to be >95%, with an intervening distance of <30 kb.
For runs of five or more markers, the fraction of informative pairs
in strong LD was required to be >95%, and markers were allowed
to span any distance.
[0081] SNP genotypes from Gabriel et al. (2002) were used for
comparison of haplotype block structure. As the density of coverage
was different between these two studies, 20 data sets were derived
from the Gabriel et al. (2002) data by randomly removing markers to
achieve the same average spacing and spacing distribution. Since
there were two existing 100-kb gaps in the SNP coverage described
herein, owing to a lack of available SNPs to type near FLOT1 and
DQB1, comparison was done by segmenting the MHC into three parts at
these gaps.
[0082] Phase Inference for Extended-Haplotype-Homozygosity
Analysis
[0083] Initial SNP, HLA, TAP, and microsatellite chromosomal
phasing was done, on the basis of segregation analysis, using the
Genehunter program (Kruglyak et al. 1996). The bulk of
genotypes--91.6% of SNP genotypes and 95% of HLA, TAP, and
microsatellite genotypes--were phased with family information.
Apart from initial phasing with family information, HLA, TAP, and
microsatellite genotypes were not phased further, and the 5% of
genotypes that were indeterminate were considered "ambiguous" in
further analyses. Further haplotype inference of SNP genotyping
data was performed with a procedure that is based on a probability
model for haplotypes proposed elsewhere (Fearnhead and Donnelly
2001). This model can be regarded as a refinement that allows for
recombination of the model used in the well-known program, PHASE
(Stephens et al. 2001). Both unphased and missing SNP data were
inferred in this manner. Since a dense set of markers were used,
and most markers are in strong LD with several other markers, the
phasing unlikely introduced serious bias into the results.
[0084] Extended-Haplotype-Homozygosity Analysis
[0085] Extended-haplotype-homozygosity (EHH) analysis was
performed, as described elsewhere (Sabeti et al. 2002), for each
haplotype block, microsatellite, HLA, and TAP allele, with cM
estimations used as distance. Grandparental chromosomes from all
families were analyzed. However, some microsatellite types (D6S258,
D6S2840, D6S2814, D6S2793, D6S1666, D6S1701, D6S1560, and D6S1542)
were not determined for five of these families (1346, 1345, 1420,
1350, and 13292). Rather than infer genotypes, these genotypes were
left as "null calls." As mentioned above, 5% of microsatellite,
HLA, and TAP genotypes could not be phased with family information.
Since EHH is a cumulative statistic, these heterozygotes and
missing data are predicted to result in a conservative estimate of
EHH values.
[0086] Outlying variants, depicted in FIGS. 3A-3C, were chosen on
the basis of two criteria designed to pick alleles with high EHH
values for their frequency class. First, as a simple approximation
of the distribution, scores were ranked by EHH value times allele
frequency. Outliers had values >4.5 SDs above the mean. Second,
all variants were sorted by frequency into 5% bins. Outliers had
EHH values >=4.79 SDs above the mean for the remaining values in
that bin.
[0087] Analysis of SNP Haplotypes around HLA-A, HLA-B, HLA-C, and
HLA-DRB1
[0088] Subsequent to the initial SNP genotyping and analysis of the
entire region, additional SNP genotyping was performed near HLA-A,
HLA-B, HLA-C, and HLA-DRB1 to assess the correlation between the
HLA genotype and local SNP haplotype. Multiblock SNP haplotypes
include information from the blocks indicated in FIG. 4, as well as
that from any intervening SNPs not in those blocks. "Leave-one-out"
cross-validation was performed using the LeaveOneOut program. In
brief, a single chromosome is selected from the data set. The
remaining samples are used to build a predictor. This predictor is
then used to predict the HLA genotype of the sample that has been
removed. If the SNP haplotype occurred once, it is not considered
in the test. For each locus, prediction was performed with 106
iterations. (See the IDRG Web site for the LeaveOneOut program and
genotyping details.)
Example 2
Analysis of the MHC Region Based on the Integrated Map
[0089] Structure of LD in the HLA Genes, Compared with the Genome
at Large
[0090] Recent studies have shown that LD extends across long
segments of the genome (Daly et al. 2001; Dawson et al. 2002;
Gabriel et al. 2002; Phillips et al. 2003). Within such segments, a
small number of distinct, common patterns of sequence variation
(haplotype alleles) are observed in the general population. Between
these segments are short intervals where recombination is
apparently most active in creating assortments of these patterns
(Daly et al. 2001; Jeffreys et al. 2001; Gabriel et al. 2002).
Operationally, it is not necessary to test each variant within an
LD segment for association with disease phenotype. Rather, a small
subset of variants that identifies all common haplotype alleles
within a segment can be used.
[0091] In order to compare the LD structure in the MHC with that of
the genome as a whole, this MHC data was compared with the data set
from Gabriel et al. (2002), as this data set offers a genomewide
comparison in which the same CEPH samples were genotyped. The
empirical definition of an LD segment or "haplotype block"
described in Gabriel et al. (2002) was used, as it provides a
common measure for comparison of genomic regions (see "Materials
and Methods" section). Because the SNP coverage described herein is
less dense than that of Gabriel et al. (2002), subsets of markers
were randomly selected from the Gabriel et al. (2002) study to
create a data set with a spacing similar to that of the present
study and thus appropriate for comparison (see "Materials and
Methods" section). Given the SNP coverage used, all haplotype
blocks are not detected. At this density, only 25% of the MHC and
14.5% of the Gabriel et al. (2002) data set is found to lie in
blocks, compared with 85% when using the full density in the
Gabriel et al. (2002) data set.
[0092] This analysis shows that that LD extends over greater
physical distances in the MHC than elsewhere in the genome (FIG.
2A). Seventeen LD segments were identified in the region that meet
the criteria of haplotype blocks (Gabriel et al. 2002) (FIGS.
1A-1E). These MHC blocks are longer, on average, in physical
distance than those found in the rest of the genome, although this
finding does not reach significance, likely because of the small
sample size (average length of 31.1 kb vs. 22.3 kb) (FIG. 2B).
[0093] Despite being longer in physical distance, haplotype blocks
in the MHC are actually shorter, in terms of genetic distance. The
average recombination rate in the MHC is 0.49 cM/Mb, versus 0.81
cM/Mb in the genome as a whole (Cullen et al. 2002; Kong et al.
2002). Given this difference in recombination rate, it was found
that blocks in the MHC have an average length of 0.012 cM, whereas
the average is 0.017 cM for the genomewide control data set
(significance not tested) (FIG. 2C). Furthermore, the distribution
of recombination across the region correlates well with most of the
long blocks (FIG. 1, asterisks) in the region. Six of the seven
largest blocks (>=75 kb) lie in areas where recombination rate
is well below the genome average of 0.81 cM/Mb. Moreover, five of
these blocks lie in regions where the recombination rate is below
the MHC regional average of 0.49 cM/Mb. The remaining large block
falls into a region where the rate is 0.83 cM/Mb. This leads to a
conclusion that the extent of LD in the MHC is longer in physical
distance but not in genetic distance than elsewhere in the
genome.
[0094] Extended-Haplotype Analysis
[0095] This work looked for alleles of haplotype blocks,
microsatellites, or classical HLA genes that occur on haplotypes
that extend across multiple blocks. Such so called "extended
haplotypes" are believed to represent a common feature of the MHC
(Alper et al. 1992). To analyze the long-range structure of the
region, EHH analysis was used, which determines the length of the
chromosomal haplotypes that extend from a specific allele at a
particular locus (Sabeti et al. 2002). High-frequency, extended
haplotypes may result from positive selection or haplotype-specific
recombination suppression. Positive selection brings rare alleles
to higher frequency in relatively few generations, thus affording
fewer opportunities for recombination events to separate an allele
from its original chromosomal context. Alternatively,
haplotype-specific recombinational suppression may result in
high-frequency, extended haplotypes by reducing the number of
recombination events a given haplotype will undergo. Since there is
a detailed sperm-typing recombination map of the region, this was
used to control for positional variation in average recombination
rates that would artificially affect the length of haplotypes.
Utilizing the integrated haplotype map, the entire MHC was scanned,
using each HLA gene, TAP gene, microsatellite, and haplotype block
as an independent locus from which to determine EHH values,
assessing every allele from a total of 46 loci.
[0096] The 50 regions in the Gabriel et al. (2002) data set each
span only 250 kb and are, therefore, not long enough to serve as a
suitable control data set for this analysis. Thus, the EHH values
of haplotype, microsatellite, and gene alleles within the MHC data
set were compared with each other and allelic variants that are
outliers were identified, on the basis of statistical rank of the
EHH value at 0.25 cM, relative to allele frequency (see "Materials
and Methods" section) (FIG. 3A). Nine alleles were identified that
map onto three different extended haplotypes (FIG. 3B). It is
striking that six of these nine variants map to a single multigene
haplotype (HLAC*0702-D6S2793*244-DRB1*1501-DQA1*0102-DQB1*0602--
D6S2876*11 [hereafter referred to as "DR2"]). Every element in the
DR2 haplotype has an EHH value at least 4.8 SDs above the mean EHH
for other variants with the same allele frequency. Two of the
remaining outlying alleles map to a single haplotype
(D6S2840*219-C*0701), and the last outlying allele is DRB1*1101. As
noted above, there are at least two possible underlying causes for
these extended haplotypes. One possibility is that a variant on the
haplotype has experienced recent positive selection. It is
interesting that each of the three extended haplotypes has been
implicated elsewhere in autoimmune disease (Thorsby 1997; Klein and
Sato 2000). The DR2 haplotype is associated with systemic lupus
erythematosus (SLE [MIM 152700]) and multiple sclerosis (MS [MIM
126200]) susceptibility, and it is protective for type I diabetes
(IDDM [MIM 222100]) (Thorsby 1997; Chataway et al. 1998; Haines et
al. 1998; Barcellos et al. 2002). DRB1*1101 is associated with
pemphigoid vulgaris, and D6S2840*219-C*0701 is associated with
autoimmune diabetes (MIM 275000) and thyroid disease (MIM 140300)
(Drouet et al. 1998; Price et al. 1999; Okazaki et al. 2000). Thus,
these three haplotypes appear to have functional consequences for
the human immune system. Although these haplotypes are associated
with autoimmune diseases at present, it is possible that, under
certain conditions, these functional differences were (and perhaps
still are) beneficial for disease resistance and, therefore, may
have undergone positive selection in the past.
[0097] The other possibility is that these extended haplotypes are
subject to allele-specific recombination suppression. By examining
the individual recombination rates used to construct the
recombination map, it is observed that, of the 12 individuals
examined, the single individual bearing DRB1*1501 showed many fewer
recombination events across the MHC than did the others, although
this difference did not significantly deviate from the mean. This
suggests that allele-specific recombination suppression could be a
possibility in this case. Further sperm typing of additional
individuals bearing each of these extended haplotypes should
resolve whether the underlying cause of this extended haplotype is
haplotype-specific recombinational suppression or whether recent
positive selection is more likely.
[0098] Common Patterns of Sequence Variation in the MHC in Regions
Between the Classical HLA Loci
[0099] Next, the haplotype block variation in the MHC was compared
with the rest of the genome. With the initial coverage, blocks that
spanned classical loci were not identified. These blocks have the
same number of common patterns of sequence variation (haplotype
alleles) as found in other regions of the genome (3.9 vs. 4.1 for
blocks with five or more markers) (FIG. 1D). Furthermore, the same
percentage of rare haplotype alleles in both data sets (3%) is
seen, which indicates that the MHC, aside from the classical loci,
does not appear to have an excess of rare haplotype variants
detectable at the current marker density. The observation that the
diversity of haplotypes outside the classical loci is typical of
the rest of the genome is perhaps surprising, given the high level
of variation at the classical HLA genes.
[0100] Common Variation in Regions Spanning the Classical HLA
Loci
[0101] The SNP haplotype diversity were separately analyzed in
regions spanning the classical HLA genes (but outside the highly
variable exons) to understand how this variation is structured. For
this purpose, it was necessary to increase the density of SNP
coverage by three- to five-fold around the four HLA genes chosen
for analysis, HLA-A, HLA-B, HLA-C, and HLA-DRB1. One motivating
question in this analysis was whether SNP haplotypes spanning
classical HLA loci contained enough information to predict HLA
alleles. If so, it might be possible to use high-throughput SNP
genotyping as a first-pass surrogate for traditional HLA gene
molecular typing (e.g., probe-based typing or direct sequencing) in
disease association studies. For one of these classical genes,
HLA-A, a single 7-SNP haplotype block spanning the locus was
identified. This 7-SNP HLA-A block has only six common variants,
and those are predictive of the correct HLA-A allele 66.2% of the
time, as shown by cross-validation analysis (LeaveOneOut [see the
"Materials and Methods" section]). To capture more of the variation
at this locus, the genotype information for a neighboring block was
included, and the SNP haplotypes that comprised the combinations of
alleles of these two blocks were examined. The success of
prediction improved from 66.2% to 82.6% of all HLA-A alleles
present.
[0102] Using such multiblock haplotypes for all four classical HLA
loci studied, multiblock SNP haplotypes can act as surrogate
markers for HLA alleles. For example, the HLA-A*0101 allele occurs
on the "G" SNP haplotype (comprising the haplotype alleles of two
blocks) 92% of the time (FIG. 4A), and the "G" SNP haplotype
correlates to HLA-A*010195.6% of the time (FIG. 4B).
Cross-validation analysis was used to estimate the success rate of
prediction. Even with the current coverage, HLA alleles can be
accurately predicted by SNP haplotype 75%/-84% of the time (HLA-A:
82.6%; HLA-B: 79.8%; HLA-C, 84.3%; and HLA-DRB1: 75.0%).
Considering only haplotypes bearing common HLA alleles (allele
frequency 15%), predictions are accurate at a higher rate (HLA-A:
96.2%, HLA-B: 98.8%; HLA-C, 96.0%; and HLA-DRB1: 82.2%) was found,
which suggests that the bulk of the prediction failures reflect an
inability to predict low-frequency variants. These data suggest
that two elements are needed to improve the predictive power: (1) a
larger data set, which would increase the numbers of observations
of rare HLA variants, and (2) increased marker density that would
provide additional SNP haplotype information, as evidenced by the
case of HLA-A above.
[0103] Electronic-Database Information
[0104] URLs for data presented herein are as follows and are
incorporated herein by reference:
[0105] Coriell Institute, http://locus.umdnj.edu/ccr/
[0106] dbSNP, http://www.ncbi.nlm.nih.gov/SNP/
[0107] IDRG, http://www.genome.wi.mit.edu/mpg/idrg/ (for
supplementary "Materials and Methods" information and pairwise D'
analysis for 201 reliable, polymorphic SNP assays in 18
multigenerational European CEPH families);
[0108] http://www.broad.mit.edu/mpg/idrg/projects/hla.htm.
[0109] Online Mendelian Inheritance in Man (OMIM),
http://www.ncbi.nlm.nih- .gov/Omim/ (for SLE, MS, IDDM, Hashimoto
thyroiditis, Graves disease).
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[0139] All references cited herein are incorporated by reference in
their entirety.
[0140] While this invention has been particularly shown and
described with references to preferred embodiments thereof, it will
be understood by those skilled in the art that various changes in
form and details may be made therein without departing from the
scope of the invention encompassed by the appended claims.
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References